Transient Stimulation with Psychoplastogens Is Sufficient to Initiate Neuronal Growth

Psychedelics (psychoplastogens) promote cortical neuron growth (increased plasticity). This study (in-vitro/cell cultures) shows that ketamine and LSD promote plasticity via TrkB (initial) and mTOR and AMPA receptor activation (sustained).

Abstract

“Cortical neuron atrophy is a hallmark of depression and includes neurite retraction, dendritic spine loss, and decreased synaptic density. Psychoplastogens, small molecules capable of rapidly promoting cortical neuron growth, have been hypothesized to produce long-lasting positive effects on behavior by rectifying these deleterious structural and functional changes. Here we demonstrate that ketamine and LSD, psychoplastogens from two structurally distinct chemical classes, promote sustained growth of cortical neurons after only short periods of stimulation. Furthermore, we show that psychoplastogen-induced cortical neuron growth can be divided into two distinct epochs: an initial stimulation phase requiring TrkB activation and a growth period involving sustained mTOR and AMPA receptor activation. Our results provide important temporal details concerning the molecular mechanisms by which next-generation antidepressants produce persistent changes in cortical neuron structure, and they suggest that rapidly excreted psychoplastogens might still be effective neurotherapeutics with unique advantages over compounds like ketamine and LSD.”

Authors: Calvin Ly , Alexandra C. Greb, Maxemiliano V. Vargas, Whitney C. Duim, Ana Cristina G. Grodzki, Pamela J. Lein & David E. Olson

Summary

Current treatments for depression are only moderately effective and are associated with a number of side effects. A better understanding of depression pathophysiology is necessary to rationally devise more effective therapeutics.

Recent studies have shown that depression results from deleterious structural and functional changes in key brain circuits. Psychoplastogens, such as ketamine, scopolamine, and serotonergic psychedelics, are the leading edge of antidepressant neurotherapeutics.

Induced plasticity has been proposed as a potential unifying mechanism to explain the efficacy of antidepressants from different chemical classes. Psychoplastogens produce more rapid changes in cortical neuron structure.

We demonstrated that ketamine and LSD, two psychoplastogens from distinct chemical classes, can increase cortical neuron growth when treated for short periods of time. This finding has important implications for central nervous system drug development.

Transient stimulation with psychoplastogens increases dendritic arbor complexity, spinogenesis, and synaptogenesis in immature cortical cultures. Treatment with ketamine or LSD for 15 min followed by a 71 h growth period results in dendritic arbor complexity comparable to what was previously observed using a 72 h treatment paradigm.

We determined that transient psychoplastogen stimulation of cortical neurons was sufficient to induce dendritic spine growth. LSD produced effects similar to those produced by BDNF, while ketamine produced more modest changes.

We performed colocalization experiments to determine the effect of transient psychoplastogen stimulation on synapse formation. The results show that psychoplastogen-induced increases in synapse density mirrored changes in spine density, with a 6 h stimulation/18 h growth period producing the greatest effects.

BDNF increased cortical structural plasticity at 50 ng/mL, but did not increase neuronal growth at higher concentrations. A psychoplastogen with BDNF-like synaptogenic efficacy has yet to be discovered.

Psychoplastogens induce cortical neuron growth by activating AMPA receptors and mTOR. DNQX, an AMPA receptor blocker, blocks both psychoplastogen-induced neuronal growth and the mTOR signaling pathway that leads to BDNF secretion, TrkB stimulation, and ultimately mTOR activation.

We hypothesized that AMPA receptor activation would lead to BDNF secretion, ultimately stimulating TrkB receptors, but this did not appear to be the case. Instead, blocking mTOR was sufficient to abrogate neuronal growth.

A psychoplastogen induces cortical neuron growth by activating AMPA receptors. Stimulation of cortical cultures with KCl did not produce a growth phenotype comparable to that produced by psychoplastogens.

Ketamine and other psychoplastogens activate AMPA receptors, TrkB, and mTOR, which lead to sustained changes in neuronal structure and behavior.

Psychoplastogens are well-known to produce effects in humans and rodents that persist long after the drug has been cleared from the body. These effects are mediated by sustained AMPA receptor and mTOR activation, which leads to glutamate release and sustained AMPA receptor activation.

Though ketamine and LSD have different primary targets, it is possible that they share an unidentified common target responsible for their psychoplastogenic effects. This hypothesis is supported by the fact that both compounds increase glutamate secretion through their respective primary targets.

Currently, it is unclear how long psychoplastogeninduced plasticity lasts, but ketamine can increase dendritic spine formation for up to 2 weeks in rodents, and psilocybin can produce antidepressant-like behavioral effects for up to 1 week.

Here, we have shown that psychoplastogens activate positive autoregulatory feedback loops leading to sustained neuronal growth. These compounds might be useful as a nonhallucinogenic alternative to traditional antidepressants, as they can be used infrequently and would have fewer side effects.

Drugs were purchased from commercial sources such as (+)-lysergic acid diethylamide (+)-tartrate (2:1) (LSD), ketamine hydrochloride (KET), (8-OH-DPAT, Sigma), brain-derived neurotrophic factor (BDNF), ANA-12, DNQX, and rapamycin.

Primary cortical cell cultures were prepared as described previously28. Cells were plated on poly-D-lysine-coated plates at specific densities depending on the experiment, and were maintained at 37 °C under an atmosphere containing 5% CO2.

In the dendritogenesis experiments, cells were treated with psychoplastogens or BDNF for short periods of time prior to exchanging the media for replacement media devoid of psychoplastogen/BDNF.

Spinogenesis experiments were performed as previously described28 with the exception that cells were treated with psychoplastogens for short periods of time prior to being fixed.

Synaptogenesis experiments were performed using 96-well plates coated with poly-D-lysine and a density of 15 000 cells per well. Cells were treated with drugs on DIV19 and then fixed with a 4% PFA solution for 20 min at room temperature. Cells were washed two times with dPBS, permeabilized with Triton X-100, blocked with antibody diluting buffer, and then incubated overnight at 4 °C with gentle shaking in antibody diluting buffer containing chicken anti-MAP2, guinea pig anti-vGLUT1, and mouse anti-PSD-95 antibodies. Images were obtained using a Molecular Devices ImageXpress Micro XLS Widefield High-Content Analysis System at 9 sites per well using 40 magnification. Analysis was performed using MetaXpress software, and the representative images shown in Figure 3B were prepared by adjusting brightness/contrast evenly across all images.

Using ImageJ and the Image Calculator, colocalization was detected between vGLUT1 and PSD-95, and a composite was created that was falsely colored to distinguish the two channels.

■ ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health, an NIH training grant, and a UC Davis Provost’s Undergraduate Fellowship. Lysergic acid diethylamide was generously provided by the NIDA Drug Supply Program.