Atrophy of neurons in the prefrontal cortex (PFC) plays a key role in the pathophysiology of depression and related disorders. The ability to promote both structural and functional plasticity in the PFC has been hypothesized to underlie the fast-acting antidepressant properties of the dissociative anesthetic ketamine. Here, we report that, like ketamine, serotonergic psychedelics are capable of robustly increasing neuritogenesis and/or spinogenesis both in vitro and in vivo. These changes in neuronal structure are accompanied by increased synapse number and function, as measured by fluorescence microscopy and electrophysiology. The structural changes induced by psychedelics appear to result from stimulation of the TrkB, mTOR, and 5-HT2A signaling pathways and could possibly explain the clinical effectiveness of these compounds. Our results underscore the therapeutic potential of psychedelics and, importantly, identify several lead scaffolds for medicinal chemistry efforts focused on developing plasticity-promoting compounds as safe, effective, and fast-acting treatments for depression and related disorders.

Because of the similarities between classical serotonergic psychedelics and ketamine in both preclinical models and clinical studies, we reasoned that their therapeutic effects might result from a shared ability to promote structural and functional neural plasticity in cortical neurons. Here, we report that serotonergic psychedelics and entactogens from a variety of chemical classes (e.g., amphetamine, tryptamine, and ergoline) display plasticity-promoting properties comparable to or greater than ketamine. Like ketamine, these compounds stimulate structural plasticity by activating the mammalian target of rapamycin (mTOR). To classify the growing number of compounds capable of rapidly promoting induced plasticity (), we introduce the term “psychoplastogen,” from the Greek roots psych- (mind), -plast (molded), and -gen (producing). Our work strengthens the growing body of literature indicating that psychoplastogens capable of promoting plasticity in the PFC might have value as fast-acting antidepressants and anxiolytics with efficacy in treatment-resistant populations and suggests that it may be possible to use classical psychedelics as lead structures for identifying safer alternatives.

Like ketamine, serotonergic psychedelics and entactogens have demonstrated rapid and long-lasting antidepressant and anxiolytic effects in the clinic after a single dose (), including in treatment-resistant populations (). In fact, there have been numerous clinical trials in the past 30 years examining the therapeutic effects of these drugs (), with 3,4-methylenedioxymethamphetamine (MDMA) recently receiving the “breakthrough therapy” designation by the Food and Drug Administration for treating PTSD. Furthermore, classical psychedelics and entactogens produce antidepressant and anxiolytic responses in rodent behavioral tests, such as the forced swim test () and fear extinction learning (), paradigms for which ketamine has also been shown to be effective (). Despite the promising antidepressant, anxiolytic, and anti-addictive properties of serotonergic psychedelics, their therapeutic mechanism of action remains poorly understood, and concerns about safety have severely limited their clinical usefulness.

Antidepressive, anxiolytic, and antiaddictive effects of ayahuasca, psilocybin and lysergic acid diethylamide (LSD): a systematic review of clinical trials published in the last 25 years.

The safety and efficacy of +/-3,4-methylenedioxymethamphetamine-assisted psychotherapy in subjects with chronic, treatment-resistant posttraumatic stress disorder: the first randomized controlled pilot study.

Ketamine has demonstrated remarkable clinical potential as a fast-acting antidepressant (), even exhibiting efficacy in treatment-resistant populations (). Additionally, it has shown promise for treating PTSD () and heroin addiction (). Animal models suggest that its therapeutic effects stem from its ability to promote the growth of dendritic spines, increase the synthesis of synaptic proteins, and strengthen synaptic responses ().

Neuropsychiatric diseases, including mood and anxiety disorders, are some of the leading causes of disability worldwide and place an enormous economic burden on society (). Approximately one-third of patients will not respond to current antidepressant drugs, and those who do will usually require at least 2–4 weeks of treatment before they experience any beneficial effects (). Depression, post-traumatic stress disorder (PTSD), and addiction share common neural circuitry () and have high comorbidity (). A preponderance of evidence from a combination of human imaging, postmortem studies, and animal models suggests that atrophy of neurons in the prefrontal cortex (PFC) plays a key role in the pathophysiology of depression and related disorders and is precipitated and/or exacerbated by stress (). These structural changes, such as the retraction of neurites, loss of dendritic spines, and elimination of synapses, can potentially be counteracted by compounds capable of promoting structural and functional neural plasticity in the PFC (), providing a general solution to treating all of these related diseases. However, only a relatively small number of compounds capable of promoting plasticity in the PFC have been identified so far, each with significant drawbacks (). Of these, the dissociative anesthetic ketamine has shown the most promise, revitalizing the field of molecular psychiatry in recent years.

Global burden of disease attributable to mental and substance use disorders: findings from the Global Burden of Disease Study 2010.

As a final note, the concentration responses of most psychoplastogens had Hill slopes that deviated from 1.0 ( Figure S3 ), implying polypharmacology. Because psychedelics have relatively high affinities for 5-HT2A receptors, it is likely that the effects of psychedelics are mediated primarily through 5-HT2A receptors at low concentrations and modulated by other targets at high concentrations. Interestingly, the concentration response of DMT was the only one to exhibit a Hill slope greater than 1.0, indicating some form of cooperativity.

These initial experiments were performed using doses of psychoplastogens that produced maximal effects on structural plasticity (circa 10 μM) in combination with a 10-fold excess of ketanserin (100 μM). At these concentrations, we could not rule out the possibility of other receptors contributing to the antagonistic effects of ketanserin. Therefore, we treated cultured cortical neurons with a significantly lower dose of LSD (10 nM) and attempted to block its ability to promote neurite outgrowth using increasing doses of ketanserin ( Figure 6 H). We found that ketanserin blocks the psychoplastogenic effects of LSD by ∼50% when treated at 10 nM. This is consistent with the fact that the binding affinities of ketanserin and LSD for the 5-HT2A receptor are roughly equivalent (low nanomolar). Increasing the concentration of ketanserin to 100 nM, 10-fold higher than the concentration of LSD used in this experiment, completely prevented LSD-induced neuritogenesis. At 100 nM, ketanserin is relatively selective for the 5-HT2A receptor, although, at this concentration, we cannot rule out the possible involvement of 5-HT2C, adrenergic, or histamine receptors.

Finally, we sought to determine whether the 5-HT2A receptor played any role in the plasticity-promoting effects of DOI, DMT, and LSD because this receptor is known to be primarily responsible for the hallucinogenic effects of classical psychedelics (). Furthermore, the psychoplastogenic potencies of these and related compounds correlate well with their 5-HT2A receptor affinities ( Figure S3 ) (i.e., a higher 5-HT2A binding affinity generally predicts more potent psychoplastogenic effects). Control experiments demonstrated that 5-HT2A receptors were expressed on cultured rat cortical neurons at both 6 days in vitro (DIV6) and DIV19 ( Figure 6 A). Next we found that co-treatment with ketanserin, a selective 5-HT2A antagonist, completely abrogated the ability of DMT, LSD, and DOI to promote both neuritogenesis and spinogenesis ( Figures 6 B–6F). Ketanserin was also able to block the effects of psilocin as well as the non-classical psychedelic noribogaine and enactogen MDMA ( Figure 6 G).

(C–E) Compound-induced increases in the AUC of the Sholl plots (C), the N max of the Sholl plots (D), and the number of dendritic branches (E) are completely blocked by ketanserin (n = 10–11 neurons, DIV6).

(B) The effects of psychedelics on increasing dendritic arbor complexity are blocked by co-treating with ketanserin, a selective antagonist of 5-HT2A receptors, as measured by Sholl analysis of cultured cortical neurons (DIV6).

Activation of TrkB is known to promote signaling through mTOR (), which plays a key role in structural plasticity (), the production of proteins necessary for synaptogenesis (), and the effects of ketamine (). Treatment with rapamycin, an mTOR inhibitor, completely blocked psychedelic-induced neuritogenesis ( Figure 5 ), thus confirming that mTOR activation plays a role in the plasticity-promoting effects of classical serotonergic psychedelics.

(A–D) The effects of psychedelics on dendritic arbor complexity are blocked by rapamycin, an inhibitor of mTOR, as measured by Sholl analysis of cultured cortical neurons (A) (DIV6). Compound-induced increases in the AUC of the Sholl plots (B), the N max of the Sholl plots (C), and the number of dendritic branches (D) are completely blocked by rapamycin (n = 9–12 neurons).

The role of BDNF in both neuritogenesis and spinogenesis is well known (), and several reports suggest that psychedelics are capable of increasing levels of neurotrophic factors (). Therefore, we treated cortical neurons with BDNF, DOI, and a combination of the two to see whether they had any additive or synergistic effects. Dose-response studies using recombinant BDNF ( Figures 3 A–3C) revealed that a 50 ng/mL treatment increased neuritogenesis to a comparable extent as DOI (10 μM). Moreover, a combination of the two did not confer any added benefit, suggesting that they operate through a related mechanism ( Figures 3 D–3F). Next, we treated cortical neurons with DOI, DMT, and LSD for 24 hr before measuring BDNF gene and protein expression using droplet digital PCR (ddPCR) and ELISA, respectively. Although psychedelics did not increase the expression of BDNF transcript ( Figure 3 G), they did result in a 2-fold increase in BDNF protein levels, although this effect was not statistically significant ( Figure 3 H). When cortical cultures were co-treated with ANA-12 (), a selective antagonist of BDNF’s high-affinity receptor TrkB, the ability of psychedelics or BDNF to stimulate neuritogenesis and spinogenesis was completely blocked ( Figure 4 ).

(A–D) The effects of psychedelics on dendritic arbor complexity are blocked by ANA-12, a selective inhibitor of TrkB, as measured by Sholl analysis of cultured cortical neurons (A) (DIV6). Compound-induced increases in the AUC of the Sholl plots (B), the N max of the Sholl plots (C), and the number of dendritic branches (D) are completely blocked by ANA-12 (n = 8–10 neurons).

(G and H) Cultured cortical neurons (DIV18) were treated with compounds for 24 hr, and then BDNF gene (G) and protein (H) expression was assessed via ddPCR (n = 4) and ELISA (n = 3–4), respectively.

(D) Sholl analysis (n = 5–10 neurons) demonstrating that DOI (10 μM) increases neuritogenesis to a comparable extent as recombinant BDNF (50 ng/mL). A combination of DOI (10 μM) and BDNF (50 ng/mL) did not have any additive or synergistic effects.

(A–C) Dose response of recombinant BDNF on neuritogenesis. AUC of the Sholl plots (A), N max of the Sholl plots (B), and total number of branches (C) of treated cortical neurons (n = 11–12 neurons per treatment, DIV6) indicate that the highest concentration of BDNF (50 ng/mL) is more effective at promoting neuritogenesis than lower concentrations (5.0 and 0.5 ng/mL).

Because the half-life of DMT is exceedingly short (∼15 min), these results confirm that structural and functional changes induced by DMT persist for hours after the compound has been cleared from the body. Moreover, they demonstrate that DMT produces functional effects on pyramidal neurons of the PFC that mirror those produced by ketamine (). Because the PFC is a key brain region involved in extinction learning (), and both ketamine and DMT have been shown to facilitate fear extinction (), our results suggest a link between the plasticity-promoting and behavioral effects of these drugs. Because fear extinction can be enhanced by increasing levels of brain-derived neurotrophic factor (BDNF) in the PFC (), and ketamine’s behavioral effects have been shown to be BDNF-dependent (), we next sought to determine the role of BDNF signaling in the plasticity-promoting effects of classical psychedelics.

Encouraged by our in vitro results, we next assessed the effects of a single intraperitoneal dose of DMT on spinogenesis in the PFC of adult rats using Golgi-Cox staining. We chose to administer a 10 mg/kg dose of DMT for three reasons. First, all available data suggested that this dose would produce hallucinogenic effects in rats with minimal safety risks (). Second, we have previously shown that a 10 mg/kg dose of DMT produces positive effects in rat behavioral tests relevant to depression and PTSD (). Finally, we wanted to directly compare the effects of DMT with ketamine, and seminal studies conducted byhad previously demonstrated that a 10 mg/kg dose of ketamine produced a robust increase in dendritic spine density in the PFC of rats. We observed a significant increase in the density of dendritic spines on cortical pyramidal neurons 24 hr after dosing with DMT ( Figures 2 I and 2J). This effect was comparable with that produced by ketamine at the same dose ( Figure 2 J). Importantly, this DMT-induced increase in dendritic spine density was accompanied by functional effects. Ex vivo slice recordings revealed that both the frequency and amplitude of spontaneous excitatory postsynaptic currents (EPSCs) were increased following DMT treatment ( Figures 2 K–2M). Interestingly, 10 mg/kg and 1 mg/kg doses produced similar responses despite the fact that they are predicted to be hallucinogenic and subhallucinogenic, respectively ().

The role of 5-HT2A, 5-HT2C and mGlu2 receptors in the behavioral effects of tryptamine hallucinogens N,N-dimethyltryptamine and N,N-diisopropyltryptamine in rats and mice.

Comparison of the discriminative stimulus effects of dimethyltryptamine with different classes of psychoactive compounds in rats.

In addition to dendritic atrophy, loss of dendritic spines is a hallmark of depression and other neuropsychiatric disorders (), so we next assessed the effects of psychedelics on spinogenesis. We treated mature rat cortical cultures for 24 hr with DOI, DMT, and LSD as representative compounds from the amphetamine, tryptamine, and ergoline classes of psychedelics, respectively. All three compounds increased the number of dendritic spines per unit length, as measured by super-resolution structured illumination microscopy (SIM) ( Figures 2 A, 2B, and S6 ), with LSD nearly doubling the number of spines per 10 μm. Additionally, treatment caused a shift in spine morphology, favoring immature (thin and filopodium) over more mature (mushroom) spine types ( Figure 2 C). Colocalization of pre- and postsynaptic markers following treatment demonstrated that psychedelics promoted synaptogenesis by increasing the density, but not the size of synapses ( Figure 2 D–2F). This increase in synapse density was accompanied by an increase in the density of VGLUT1 puncta, but not PSD-95 puncta, following compound administration ( Figures 2 G and 2H).

p < 0.05,p < 0.01,p < 0.001,p < 0.0001, as compared to vehicle control (VEH). Data are represented as mean ± SEM. See also Figure S6

(K and L) Whole-cell voltage-clamp recordings of layer V pyramidal neurons from slices obtained 24 hr after DMT treatment (10 mg/kg and 1 mg/kg) demonstrate that DMT increases both spontaneous excitatory postsynaptic current (sEPSC) frequency (K) and amplitude (L) (n = 11–38 neurons from 3 animals).

(D) Representative images of cortical neurons (DIV19) treated for 24 hr, demonstrating that psychedelics increase synaptogenesis (green, VGLUT1; magenta, PSD-95; yellow, MAP2). White areas in the VGLUT1 + PSD-95 images indicate colocalization of pre- and postsynaptic makers and are indicated by gray arrows.

To assess the in vivo effects of classical psychedelics on neuritogenesis, we started treating Drosophila larvae during the first instar with LSD and DOI. As observed in rodent cortical cultures, both LSD and DOI significantly increased dendritic branching of class I sensory neurons; however, they did not increase the total length of the dendritic arbors ( Figures 1 J–1L). Because of the striking effects of psychedelics on the structures of immature neurons, we hypothesized that they might influence neurodevelopment. To test this, we chronically treated zebrafish embryos with compounds for 6 days immediately following dechorionation and assessed gross morphological changes and behavior. We did not observe any differences in head sizes between the treatment groups, nor did we detect any statistically significant differences in activity levels ( Figure S5 ). Next we assessed the ability of psychedelics to promote neuritogenesis in more mature neurons by starting to treat Drosophila larvae during the late second instar. Again, psychedelics increased the branching of class I neurons, although the effect was less dramatic than that observed when treatment was started during the first instar ( Figure 1 M–1O). Although different developmental stages might be more or less susceptible to the effects of psychedelics, it is also possible that the smaller effect size observed after administering compounds starting at the later time point was simply the result of treating the larvae for a shorter period of time. Regardless, it was quite surprising to observe compound-induced changes in neuronal structure after initiating treatment during the late second instar because class I neurons are stereotyped and typically possess relatively few higher-order branches (). Moreover, our results demonstrate that psychedelics can promote changes in neuronal structure across vertebrate (rats) and invertebrate (Drosophila) species, suggesting that they act through an evolutionarily conserved mechanism.

Notably, the anti-addictive alkaloid ibogaine () was the only psychedelic tested that had absolutely no effect ( Figure S4 ). This was a surprising result because we hypothesized that ibogaine’s long-lasting anti-addictive properties might result from its psychoplastogenic properties. Previous work byclearly demonstrated that ibogaine increases the expression of glial cell line-derived neurotrophic factor (GDNF) and that this plasticity-promoting protein is critical to ibogaine’s anti-addictive mechanism of action. Because several reports have suggested that noribogaine, a metabolite of ibogaine, might actually be the active compound in vivo (), we decided to test its ability to promote neuritogenesis in cultured cortical neurons. Gratifyingly, noribogaine robustly increased dendritic arbor complexity with an ECvalue comparable to ketamine ( Figure S3 ), providing additional evidence suggesting that it may be the active compound in vivo.

To establish the relative potencies and efficacies of hallucinogens and entactogens for promoting neurite outgrowth, we conducted 8-point dose-response studies ( Figure S3 ). We defined 100% and 0% efficacy as the maximum number of crossings induced by ketamine (10 μM) and vehicle (0.1% DMSO), respectively. We chose the 10 μM concentration of ketamine as the upper limit because this concentration of ketamine is reached in the brain following intraperitoneal administration of an antidepressant dose in rats (). For consistency, we used this same concentration when testing the effects of psychedelics and entactogens, with DMT being the only exception. We used a maximum 90 μM concentration of DMT in our studies to more closely mimic the brain concentration of DMT in rats treated with an antidepressant dose (). In this neuritogenesis assay, ketamine’s half maximal effective concentration (EC) value was 132 nM. Surprisingly, the majority of the psychedelics and entactogens we tested exhibited significantly greater potency than ketamine, with LSD being particularly potent (EC= 0.409 nM). In fact, LSD exhibited activity across 8 orders of magnitude into the low picomolar range ( Figure S3 ).

Nearly all psychedelic compounds tested were capable of robustly promoting neuritogenesis, with comparable effects being produced by tryptamines (N,N-dimethyltryptamine [DMT] and psilocin), amphetamines (2,5-dimethoxy-4-iodoamphetamine [DOI] and MDMA), and ergolines (lysergic acid diethylamide [LSD]). As a positive control, we treated cells with 7,8-dihydroxyflavone (DHF), a psychoplastogen structurally dissimilar to classical psychedelics (), and found that it also increased dendritic arbor complexity ( Figure S2 ). This neurite outgrowth structural phenotype seems to only be induced by select compounds because serotonin and D-amphetamine, molecules that are chemically related to classical psychedelics and entactogens, exerted minimal to no effects on neuritogenesis ( Figure S2 ).

Because atrophy of cortical neurons is believed to be a contributing factor to the development of mood and anxiety disorders (), we first treated cultured cortical neurons with psychedelics from a variety of structural classes ( Figures 1 A and S1 A) and measured the resulting changes in various morphological features. Using Sholl analysis (), we observed that several psychedelics increased dendritic arbor complexity comparably to ketamine, as measured by the area under the curve of the Sholl plots as well as the maximum number of crossings ( Figures 1 B–1E and S1 B–S1E). This increase in arbor complexity appeared to result from large changes in both the number of dendritic branches and the total length of the arbors ( Figures 1 F, 1H, S1 F, and S1H). Psychedelics had a limited effect on the number of primary dendrites and did not alter the length of the longest dendrite ( Figures 1 G, 1I, S1 G, and S1I).

p < 0.05,p < 0.01,p < 0.001,p < 0.0001, as compared to vehicle control (VEH). Scale bars, 30 μm. Data are represented as mean ± SEM. See also Figures S1–S5

(M and N) Class I neurons from Drosophila larvae treated with psychedelics during the third instar display increased branching (M) but not total length of the dendritic arbor (N) (n = 3 neurons).

(J and K) Class I neurons from Drosophila larvae treated with psychedelics during the first instar display increased branching (J) but not total length of the dendritic arbor (K) (n = 3 neurons).

(F–I) Cortical neurons treated with psychedelics display an increase in the number of branches (F), the number of primary dendrites (G), and the total length of the dendritic arbor (H) but not the length of the longest dendrite (I).

Discussion

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et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Although the molecular targets of ketamine and psychedelics are different (NMDA and 5-HT2A receptors, respectively), they appear to cause similar downstream effects on structural plasticity by activating mTOR. This finding is significant because ketamine is known to be addictive whereas many classical psychedelics are not (). The exact mechanisms by which these compounds stimulate mTOR is still not entirely understood, but our data suggest that, at least for classical psychedelics, TrkB and 5-HT2A receptors are involved. Although most classical psychedelics are not considered to be addictive, there are still significant safety concerns with their use in medicine because they cause profound perceptual disturbances and still have the potential to be abused. Therefore, the identification of non-hallucinogenic analogs capable of promoting plasticity in the PFC could facilitate a paradigm shift in our approach to treating neuropsychiatric diseases. Moreover, such compounds could be critical to resolving the long-standing debate in the field concerning whether the subjective effects of psychedelics are necessary for their therapeutic effects (). Although our group is actively investigating the psychoplastogenic properties of non-hallucinogenic analogs of psychedelics, others have reported the therapeutic potential of safer structural and functional analogs of ketamine ().

Our data demonstrate that classical psychedelics from several distinct chemical classes are capable of robustly promoting the growth of both neurites and dendritic spines in vitro, in vivo, and across species. Importantly, our studies highlight the similarities between the effects of ketamine and those of classical serotonergic psychedelics, supporting the hypothesis that the clinical antidepressant and anxiolytic effects of these molecules might result from their ability to promote structural and functional plasticity in prefrontal cortical neurons. We have demonstrated that the plasticity-promoting properties of psychedelics require TrkB, mTOR, and 5-HT2A signaling, suggesting that these key signaling hubs may serve as potential targets for the development of psychoplastogens, fast-acting antidepressants, and anxiolytics. Taken together, our results suggest that psychedelics may be used as lead structures to identify next-generation neurotherapeutics with improved efficacy and safety profiles.