A Single Dose of 5-MeO-DMT Stimulates Cell Proliferation, Neuronal Survivability, Morphological and Functional Changes in Adult Mice Ventral Dentate Gyrus

This animal study (n=220) investigated the effects of 5-MeO-DMT (100 μg) on neuronal growth in the hippocampus of mice and found that a single dose increased the proliferation of neural progenitors, accelerated the maturation of newborn granule cells, and increased the complexity of the dendritic morphology.

Abstract

Introduction: The subgranular zone (SGZ) of dentate gyrus (DG) is one of the few regions in which neurogenesis is maintained throughout adulthood. It is believed that newborn neurons in this region encode temporal information about partially overlapping contextual memories. The 5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a naturally occurring compound capable of inducing a powerful psychedelic state. Recently, it has been suggested that DMT analogs may be used in the treatment of mood disorders. Due to the strong link between altered neurogenesis and mood disorders, we tested whether 5-MeO-DMT is capable of increasing DG cell proliferation.

Methods/Results: We show that a single intracerebroventricular (ICV) injection of 5-MeO-DMT increases the number of Bromodeoxyuridine (BrdU+) cells in adult mice DG. Moreover, using a transgenic animal expressing tamoxifen-dependent Cre recombinase under doublecortin promoter, we found that 5 Meo-DMT treated mice had a higher number of newborn DG Granule cells (GC). We also showed that these DG GC have more complex dendritic morphology after 5-MeO-DMT. Lastly, newborn GC treated with 5-MeO-DMT, display shorter afterhyperpolarization (AHP) potentials and higher action potential (AP) threshold compared.

Discussion: Our findings show that 5-MeO-DMT affects neurogenesis and this effect may contribute to the known antidepressant properties of DMT-derived compounds.”

Authors: Rafael V. L. da Cruz, Thiago C. Moulin, Lyvia L. Petiz & Richardson N. Leão

Summary

INTRODUCTION

Psychoactive tryptamines are found in many plants in South America, including Ayahuasca, which is used ritualistically by many syncretic churches to treat many physical and mental illnesses.

Adult neurogenesis occurs in two sites in the brain, the subgranular zone (SGZ) of the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle. It is not known whether in vivo adult neurogenesis is affected by psychoactive tryptamines.

We tested if a single dose of 5-MeO-DMT affects neurogenesis in mice. We found that the number of DCX::tdTom+ cells and dendritic trees were higher in experimental group than in control group.

5-MeO-DMT Treatment

Animals anesthetized with isoflurane received a single ICV injection of 5-MeO-DMT solution. Control groups received 1 l of 10% DMSO in saline.

BrdU Labeling, Tamoxifen Treatment, Cryopreservation and Slicing

Animals were injected with BrdU diluted in saline ten to fifteen minutes after 5-MeO-DMT or saline ICV injections, and were either sacrificed or perfused for histology or patch clamp experiments. For histology experiments, brains were removed from PFA perfused animals, postfixed in 4% PFA overnight, washed in PB 0.1 M (pH = 7.4) for 10 min, and then cryopreserved in graded sucrose solutions (10/20/30%). Forty micrometer horizontal hippocampal sections were cut in a cryostat.

BrdU Immunohistochemistry

Hippocampal slices were washed with PBS, placed in HCl 2N for 30 min at 37 C to open DNA double strand, washed again in PBS, transferred to borate buffer (pH = 8.0) at RT for 20 min, incubated overnight in primary antibody solution, and then mounted on N-propyl gallate solution mounting medium.

Clustering Analysis of BrdU+ Cells

After microscopy, images were processed by a personal MATLAB code, and a graph was created. The number of clusters was measured, and a modularity algorithm was used to determine the number of cells within each cluster.

Electrophysiology and Dendritic

Animals were anesthetized with ketamine and xylazine, then perfused with RT standard cerebrospinal fluid, decapitated, and their brains removed. They were then transferred to a vibratome chamber containing ice-cold aCSF, and then again returned to aCSF for at least 1 h at RT prior to recordings. For whole-cell patch clamp recordings, GC from DCX-CreERT2 ::tdTomlox/lox mice were identified by fluorescence (543 excitation/580 emission) and current-clamp recordings were obtained using an axopatch amplifier 200B (Molecular Devices) in whole-cell configuration using the winWCP Strathclyde Electrophysiology Software.

We recorded currents and spontaneous excitatory postsynaptic currents in hippocampal slices from DCX-CreERT2 ::tdTomlox/lox mice, and analyzed the data using WinWCP and winECP. We also analyzed dendritic morphology using ImageJ and Sholl analysis.

Statistical Analysis

All data was normal distributed, tested for normality with D’Agostino and Pearson omnibus normality test, and compared between groups with unpaired t-test. Sholl analysis of dendritic arborization was performed with two-way ANOVA and Holm-Sidak’s post hoc test.

RESULTS

5-MeO-DMT increased cell proliferation in the ventral DG of adult mice compared to saline injected controls (p = 0.0029, unpaired t-test, Figures 1A,B). We analyzed the cluster formation of BrdU+ cells in the ventral DG of control and 5-MeO-DMT injected mice. We found that the number of clusters was greater in 5-MeO-DMT treated mice than saline treated mice, and the number of cells per cluster was trending to significance.

Following the proliferation assay, we tested if the number of newborn DG GC cells was higher after ICV injection of 5-MeO-DMT. The results indicate that the 5-MeO-DMT increase survivability of newborn neurons generated within the hippocampus.

We performed whole cell patch clamp experiments on DCX-CreERT2 ::tdTomlox/lox GC to study if 5-MeO-DMT can modify electrophysiological properties of immature DG GC. Cells from 5-MeO-DMT-treated animals displayed higher AP threshold and shorter AHP potential duration. We applied current ramps to the membrane potential of 5-MeO-DMT-treated mice to elucidate differences in fast activated currents.

Young GC from 5-MeO-DMT-injected mice show a higher degree of maturation than cells from control animals, as evidenced by the higher linear regression ramp current vs. AP instantaneous in 5-MeO-DMT group.

We recorded sEPSCs from six mice treated or not with 5-MeO-DMT and found that sEPSC amplitude and frequency were increased in tdTomato+ cells of 5-MeO-DMT-treated mice when compared to controls. We then tested if 5-MeO-DMT alters morphological maturation of newborn GC.

The experimental group had a higher number of dendritic branches and more intersections in the 50 – 170 m range of distance from soma when compared to saline. This suggests that 5-MeO-DMT accelerates dendritic growth toward a morphology of a fully mature DG granule neuron.

DISCUSSION

We showed that 5-MeO-DMT increases proliferation of neural progenitors and accelerates the maturation of newborn GC. The dendritic trees of newborn neurons from 5-MeO-DMT-treated mice were significantly more complex and had a lower AP threshold and longer AHP potential compared to controls.

The increased number of BrDU+ cells indicate that more cells are entering in the S-phase of cell-cycle following 5-MeO-DMT injection, but cannot elucidate the type of progenitor cell that is being affected.

The study chose a single dose treatment to address the gap between the molecular mechanisms, subjective and hormonal effects underlying Ayahuasca acute administration to depression diagnosed patients. The study isolated the effect of the 5-MeO-DMT, without adding any bias, due to other psychoactive compounds.

The results indicate that 5-MeO-DMT increases both proliferation and survivability of granule cells in the ventral hippocampus, and also increases the number of cells that reach neuronal maturity. Hence, 5-MeO-DMT has a positive effect on adult neurogenesis differing from serotonin alone.

Young neurons from 5-MeO-DMT-treated mice show faster maturation than cells from control animals, as indicated by the morphology of dendritic trees and the larger hyperpolarizing-activated current.

The Dorsal Raphe Nucleus targets the SGZ and is a strong 5-HT2A and 5-HT2C agonist, but it also acts in other receptors (with much lower potency). Hence, specific 5HT receptors might be involved in neurogenesis modulation.

We show that a single dose of 5-MeO-DMT can increase proliferation, survivability and accelerate maturation of newborn neurons in the DG, and that this effect could explain the beneficial effects of hallucinogenic compounds in mood disorders.

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