5-MeO-DMT

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

This mouse 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.

Authors

Leão, R. N.
Lima da Cruz, R. V.
Moulin, T. C.

Published

September 4, 2018

Frontiers in Molecular Neuroscience

individual Study

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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.

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

Introduction

Psychoactive tryptamines such as N,N-dimethyltryptamine (DMT) and its methoxy analogue 5-MeO-DMT act on serotonergic receptors and are present in several South American plants and traditional preparations such as ayahuasca. Previous work links altered adult hippocampal neurogenesis to mood disorders and to the action of some antidepressant treatments, and in vitro evidence suggests that certain alkaloids present in ayahuasca can stimulate neurogenesis. However, whether in vivo adult neurogenesis is affected by psychoactive tryptamines—and specifically by 5-MeO-DMT—remains unclear. Huang and colleagues set out to test whether a single intracerebroventricular (ICV) dose of 5-MeO-DMT alters dentate gyrus (DG) neurogenesis in adult mice. The study assessed cell proliferation using BrdU labelling, quantified newborn neurons with an inducible DCX-driven fluorescent reporter line, examined clustering of proliferating cells, and evaluated electrophysiological and dendritic-morphological maturation of newborn granule cells to determine whether 5-MeO-DMT affects proliferation, survivability and maturation of DG neurons.

Methods

The study followed institutional animal-care approvals. Adult C57BL/6J mice and DCX-CreER T2 ::tdTom lox/lox transgenic mice (both sexes, aged 55–70 days) were housed on a 12 h light/dark cycle with food and water ad libitum. Treatment: under isoflurane anaesthesia animals received a single ICV injection (stereotaxic coordinates reported) of 1 µL containing 100 µg 5-MeO-DMT in 10% DMSO/90% saline; control animals received 1 µL 10% DMSO in saline. Ten to fifteen minutes after ICV injection animals were administered BrdU (50 mg/kg, intraperitoneal) to label S-phase cells. For proliferation assays, mice were sacrificed 12 h after BrdU injection. In DCX-CreER T2 ::tdTom lox/lox mice tamoxifen (100 µg/g/day, IP) was given starting 3 days after ICV injection to induce reporter expression; these animals were subsequently processed either for histology or for electrophysiology and morphology studies. Tissue processing and histology: perfusion with paraformaldehyde, cryopreservation through graded sucrose and horizontal sectioning at 40 µm were performed. Eight spaced slices per animal containing ventral hippocampi were selected for BrdU immunohistochemistry; BrdU+ cells were manually counted by a blinded experimenter. DCX::tdTomato+ cells were counted in a single hemisphere for the transgenic line because the contralateral hemisphere was reserved for electrophysiology. Cluster analysis: digital images were processed with custom MATLAB code treating each BrdU+ cell as a node; edges were created for intercellular distances <25 µm and Girvan-Newman modularity was used to identify clusters, defined as groups of 1–5 cells, with counts of clusters and cells per cluster extracted. Electrophysiology and morphology: 300 µm hippocampal slices were prepared in protective solutions and recorded in standard aCSF. DCX::tdTomato+ granule cells were targeted for whole-cell patch clamp. Current-clamp protocols included 100 ms current steps (-100 pA to 400 pA, 50 pA increments) and a 1.5 s ramp (-50 pA to 200 pA). Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in voltage clamp at -60 mV and detected using a template-based Matlab routine. Some slices were fixed for confocal imaging and Sholl analysis; dendritic tracing used Simple Neurite Tracer in ImageJ to quantify branch number and intersections. Statistics: normality was assessed with the D'Agostino and Pearson test. Group comparisons used unpaired t-tests for most measures; Sholl analysis employed two-way ANOVA with Holm–Sidak post hoc comparisons at 10 µm concentric intervals. Data are reported as mean ± SEM and sample sizes are reported in the Results.

Results

BrdU proliferation assay: mice given a single ICV dose of 5-MeO-DMT had more BrdU+ cells in the ventral dentate gyrus than saline controls (saline 155.4 ± 21.71 BrdU+ cells/animal, n = 5; 5-MeO-DMT 352.6 ± 41.48, n = 5; p = 0.0029, unpaired t-test). Cluster analysis: the number of BrdU+ cell clusters per section was greater after 5-MeO-DMT (saline 5.964 ± 0.5718 clusters/section, n = 5; 5-MeO-DMT 11.7 ± 0.6974 clusters/section, n = 5; p = 0.0002), suggesting recruitment of more progenitors. The mean number of cells per cluster showed a trend toward increase with 5-MeO-DMT (saline 1.433 ± 0.1365 vs 5-MeO-DMT 1.846 ± 0.119; p = 0.0523). Newborn neuron survivability: using DCX-CreER T2 ::tdTom reporter mice, the number of DCX::tdTom+ cells in the ventral hippocampus was higher in the 5-MeO-DMT group (saline 104 ± 6.348 cells, n = 6; 5-MeO-DMT 220.3 ± 22.86 cells, n = 6; p = 0.0006), indicating increased numbers of immature neurons surviving to reporter expression. Electrophysiology: whole-cell recordings from tdTomato+ newborn granule cells showed altered intrinsic properties after 5-MeO-DMT. Action potential (AP) threshold was less negative in treated cells (saline -38.25 ± 2.03 mV, n = 8 cells/3 animals; 5-MeO-DMT -29.60 ± 2.51 mV, n = 11 cells/3 animals; p = 0.022). Afterhyperpolarization (AHP) duration was shorter in the 5-MeO-DMT group (saline 53.33 ± 12.04 ms, n = 9 cells/3 mice; 5-MeO-DMT 12.40 ± 1.302 ms, n = 8 cells/3 mice; p = 0.006). The frequency–current relationship from ramp protocols had a steeper slope after 5-MeO-DMT (controls 0.11 ± 0.01 Hz/pA, n = 8 cells/3 animals; 5-MeO-DMT 0.17 ± 0.03 Hz/pA, n = 6 cells/3 animals; p = 0.03). In random sampling, 3/20 tdTom+ cells from saline-treated animals failed to fire action potentials, whereas all 16 cells from 5-MeO-DMT-treated animals fired (p < 0.0001, z test), consistent with accelerated functional maturation. Synaptic inputs: sEPSC amplitude was larger in newborn cells from 5-MeO-DMT-treated mice (57.15 ± 4.68 pA vs 37.72 ± 6.60 pA in controls; p = 0.03), and sEPSC frequency was substantially increased (1.35 ± 0.22 Hz vs 0.35 ± 0.07 Hz; p = 0.002), indicating enhanced excitatory synaptic drive. Morphology: dendritic complexity of tdTomato+ newborn neurons was greater after 5-MeO-DMT. The mean number of dendritic branches per cell increased (saline 7.15 ± 0.4247 branches, n = 20 cells from six mice; 5-MeO-DMT 12.6 ± 0.916 branches, n = 15 cells from six mice; p = 0.0001). Sholl analysis revealed a higher number of dendritic intersections in treated neurons across the 50–170 µm radius range from the soma, consistent with more elaborate dendritic arbors. (The extracted Sholl values are reported in the source text for multiple radii but the presented extraction is truncated before all radii are listed.)

Discussion

Huang and colleagues interpret their data as demonstrating that a single ICV dose of 5-MeO-DMT increases proliferation of neural progenitors in the adult dentate gyrus and promotes greater survivability and accelerated maturation of newborn granule cells. The increase in BrdU+ cells suggests more cells entering S phase, while the rise in DCX::tdTom+ cell counts indicates more immature neurons reaching later developmental stages. Electrophysiological and morphological measures—shorter AHPs, higher AP thresholds, greater firing responsiveness, increased sEPSC amplitude and frequency, and more complex dendritic arbors—are presented as convergent evidence of faster functional and structural maturation in newborn neurons after 5-MeO-DMT. The authors acknowledge that BrdU labelling alone cannot identify the specific progenitor subtype affected and note that markers such as GFAP, nestin and Sox2, or proliferation markers like Ki67 or MCM2, would be needed to distinguish recruitment from S-phase lengthening. They explain the choice of a single ICV injection: delivering 5-MeO-DMT centrally avoids peripheral monoamine oxidase degradation and isolates 5-MeO-DMT’s effects from other compounds present in ayahuasca preparations, such as harmine, which itself can affect neurogenesis. Huang and colleagues emphasise that while 5-MeO-DMT is a potent agonist at 5-HT2A and 5-HT2C (and acts at other receptors with lower potency), the present data do not identify the receptor mechanisms involved; selective agonist/antagonist studies are needed to dissect molecular pathways. Finally, the authors place their findings in the context of prior work linking serotonergic hallucinogens and antidepressant effects, proposing that modulation of adult neurogenesis by 5-MeO-DMT could be one mechanism underlying beneficial effects of tryptamine-derived compounds in mood disorders. They call for further work to characterise voltage-dependent currents (for example I h ), chloride reversal potential during development, and receptor-specific contributions to the observed effects.

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INTRODUCTION

Psychoactive tryptamines are a class ofmolecules that act as a neurotransmitter in the vertebrate brain. N,N-dimethyltryptamine, (DMT) and analogues, are closely related to 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), they can be found in a great variety of plants in South America, with an even greater diversity of chemical analogs. 5-MeO-DMT is a serotonin agonist that acts in a non-selective manner in 5-HT 2A >5-HT 2C >5-HT 1A receptors. However, the N-N-DMT has been reported elsewhere to also acts in many glutamate, dopamine, and acethylcholine receptors. It would be interesting to know whether the 5-MeO-DMT have the same effect as its analogue on those receptors. The 5-MeO-DMT is analogous of the N,N-DMT, one of the main active ingredients of Ayahuasca, a millenarian decoction used as a sacrament by south American indigenous tribes, known to induce powerful hallucinogenic states when administered with monoamine oxidase inhibitors (MAOI;. At present, Ayahuasca is used by many syncretic churches ritualistically, as a way to heal many physical and mental illnesses with or without scientific knowledge about the effects. Recent studies also suggest that Ayahuasca can potentially treat recurrent depressioneven in a placebo controlled frame. Deficits in adult neurogenesis are associated with the physiopathology of depression and modulation of neurogenesis is behind the action of several antidepressants. Serotonin reuptake inhibitors, for example, rescue normal neurogenesis levels in animal models of depression. Adult neurogenesis is known to occur in two sites in the brain, the subgranular zone (SGZ) of the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle. There is some debate if SVZ neurogenesis responds or not to mood disorders and psychoactive drugsbut the effect of mood disorders in SGZ Radial glial Like cell (RGL) proliferation and neuronal survivor is prolifically described. Interestingly, alkaloids from one of the plants used in the Ayahuasca brew stimulate neurogenesis in vitro; however, it is not known whether in vivo adult neurogenesis is affected by psychoactive tryptamines. In this study we tested if a single dose of 5-MeO-DMT affects neurogenesis in mice. We found that after a single intracerebroventricular (ICV) injection of 5-MeO-DMT, cell proliferation in the DG was significantly larger in comparison to saline. Moreover, the number of DCX::tdTom+ cells are also higher for experimental group, these same DG granule cells (GC) show more complex dendritic trees when compared to control animals. Finally, we found that afterhyperpolarization (AHP) potential duration where shorter and action potential (AP) threshold higher in newborn neurons from mice treated with 5-MeO-DMT.

ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the National Council for the Control of Animal Experimentation (CONCEA) in Brazil. The protocol was approved by the local animal care institution of the Federal University of Rio Grande do Norte (Protocols 041/2014 and 015.004/2017).

ANIMALS

Adult C57BL6J and DCX-CreER T2 ::tdTom lox/lox transgenicmice from both sex aged between 55-70 days were used in this study. Animals were housed under a 12 h light/12 h dark cycle. Food and water ad libitum.

-MEO-DMT TREATMENT

Animals anesthetized with isoflurane (3%-5% L/min for induction and 1%-3% L/min for maintenance;received a single ICV injection of 1 µL 5-MeO-DMT solution (100 µg 5-MeO-DMT in 10% DMSO/90% saline) prepared freshcontrol groups received 1 µL of 10% DMSO in saline (stereotaxic coordinates: 0.3 mm AP, 1.0 mm ML and 2.8 mm DV;.

BRDU LABELING, TAMOXIFEN TREATMENT, CRYOPRESERVATION AND SLICING

Ten to fifteen minutes after 5-MeO-DMT or saline ICV injections, animals (under anesthesia) received 50 mg/kg of Bromodeoxyuridine (BrdU, Sigma) intraperitoneally (IP) diluted in saline. For proliferation assays mice were sacrificed (overdose of ketamine 130 mg/kg mixed with 8 mg/kg xylazin) and perfused with PBS followed by paraformaldehyde (PFA) 12 h after BrdU injection. To induce recombination in DCX-CreER T2 ::tdTom lox/lox animals were treated 100 µg/g/day of tamoxifen IP 3 days after ICV injections. These animals were either perfused for histology following the same slicing and freezing protocol for BrdU staining, or anesthetized and had the brains removed for patch clamp experiments (see below). For histology experiments, brains from PFA perfused animals were removed and postfixed in 4% PFA overnight. Brains were then washed in PB 0.1 M (pH = 7.4) for 10 min then immersed in graded sucrose solutions (10/20/30%) for cryopreservation, then snap frozen by immersion into -80 • C isopropyl alcohol and stored in -80 • C freezer for posterior cryosectioning. Forty micrometer horizontal hippocampal sections were cut in a cryostat (Thermo Microm HM 550) for BrdU immunohistochemistry and DCX::tdTom count. Eight slices from each animal, containing both ventral hippocampi, were gathered, spaced with 200 µm between them (every fifth slice were collected) to avoid sample the same population twice. In DCX-CreER T2 ::tdTom lox/lox cell counting were performed in a single hemisphere as the other hemisphere was used for patch clamp experiments (in order to reduce the number of animals used for the experimental purpose, in accordance with local guidelines of the the Brazilian guidelines for laboratory animal welfare).

BRDU IMMUNOHISTOCHEMISTRY

Hippocampal slices were washed with PBS (pH = 7.4) for 10 min at room temperature (RT), then placed for 30 min into HCl 2N at 37 • C to open DNA double strand, washed again in PBS, transferred to borate buffer (pH = 8.0) at RT for 20 min, then washed in PBS and incubated overnight in primary antibody solution: 10% normal goat serum (NGS; Sigma), 1:500 Rat igG anti-BrdU (Abcam) and 0.3% X-100 triton in PBS solution (Sigma). Slices were then washed in PBS for 10 min and incubated for 2 h in secondary antibody solution: 10% NGS, 1:1,000 rabbit igG anti-rat rabbit F(ab')2 Anti-Rat IgG H&L conjugated with Alexa Fluor 488 (Abcam) and 0.3% triton x-100 in PBS. Slices were subsequently washed with PBS solution and incubated in 1:2,000 Hoechst 33425 (ThermoFisher) in PBS 10 mM (nuclei staining), washed in PBS and mounted on N-propyl gallate solution mounting medium. Hippocampal slices were imaged using an epifluorescence upright microscope (ZEISS) with Stereoinvestigator software (MBF Bioscience), BrdU+ cells were manually counted in both hippocampi by an experimenter blinded for groups.

CLUSTERING ANALYSIS OF BRDU+ CELLS

After microscopy, images were processed by a personal MATLAB code, where the total number of cells and distances between them were calculated. We computed this data to generate a graph where each cell was considered a node. If the distance between two cells was less than 25 µm, an edge was created between them and its weight was increased as closer they were. We analyzed the final graph using the Girvan-Newman's modularity algorithm, Considering clusters as groups of 1-5 cells. We then measured how many clusters were formed and the number of cells within clusters.

ELECTROPHYSIOLOGY AND DENDRITIC MORPHOLOGY ANALYSIS

DCX-CreER T2 ::tdTom lox/lox animals were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (8 mg/kg) then intracardially perfused with RT standard cerebrospinal fluid (aCSF; in mM: NaCl 124; KCl, 2.5; NaH 2 PO 4 , 1.2; NaHCO 3 , 24; glucose, 12, 5; CaCl 2 , 2; MgCl 2 , 2). Animals were then decapitated and had their brains removed and then transferred to a vibratome chamber containing ice-cold aCSF, slices with 300 µm thickness were collected in the Vibratome (VT1200, Leica) and transferred to a custom designed 3d printed incubation chamber containing recover NMDG solution (in mM NMDG, 92; KCl, 2.5; NaH 2 PO 4 , 1.25; NaHCO 3 , 30; HEPES, 20; glucose, 25; thiourea, 2; sodium-ascorbate, 5; sodium-pyruvate, 3; CaCl 2 •4H 2 O, 0.5; 10 MgSO 4 •7H 2 , 10; pH controlled to 7.3-7.4 with 2N HCl solution) at 36 • C for 15 min, and then again returned to aCSF for at least 1 h at RT prior to recordings, all solutions were continually bubbled with carbogen 95% O 2 and 5% CO 2 (White-Martins;. For whole-cell patch clamp recordings the tissue was transfered to a chamber filled with Standard aCSF in a Microscope (ZEISS). Micropipettes were filled with K-gluconate solution (in mM, K-Gluconate, 145; HEPES, 10; EGTA, 1; Mg-ATP, 2; Na2-GTP, 0.3; MgCl 2 , 2; pH 7.3, 290-300 mOsm) GC from DCX-CreER T2 ::tdTom lox/lox mice were identified by fluorescence (543 excitation/580 emission). Current-clamp recordings were obtained using an axopatch amplifier 200B (Molecular Devices) in whole-cell configuration using the winWCP Strathclyde Electrophysiology Software. Two protocols were used in current clamp: 100 ms-long current steps with 50 pA increment ranging from -100 pA to 400 pA and a ramp ranging from -50 pA to 200 pA in 1500 ms. Current clamp data was analyzed using winWCP. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in voltage clamp using winECP (free run-cells held at -60 mV). To analyze sEPSCs, events were first detected using a custom Matlab (Mathworks) program ('pspAnalysis.m'). The program detects EPSCs based on a template using correlation coefficient calculated in a sliding window. The program can be downloaded from. Some hippocampal sections were fixed after slices and used for Sholl analysis. To analyze dendritic morphology, slices from DCX-CreER T2 ::tdTom lox/lox mice, obtained as above, were kept overnight in PFA 0.4% overnight and 40× -amplification pictures were taken using confocal microscopy (Zeiss) to analyze the Tomato-expressing neurons. The images in which was possible to clearly visualize the dendritic arborization were blinded selected to experimental groups. Morphometry was performed using the ImageJ plug-in Simple Neurite Tracer, extracting the number of branches and performing Sholl analysis.

STATISTICAL ANALYSIS

All data is normal distributed, tested for normality with D'Agostino and Pearson omnibus normality test. Comparisons between groups were made with unpaired t-test. For BrdU staining, cell clustering analysis and DCX-CreER T2 ::tdTom lox/lox cell counting, eight sections containing ventral hippocampus was chosen from each animal, dorsal hippocampus and the smallest portion of ventral hippocampus from each animal were excluded from analysis. For mean comparison, the total number of BrdU+ cells or DCX::tdTOM+ cells were accounted from each selected section and summed up, then, the total number per animal were used for unpaired t-test. For Sholl analysis of dendritic arborization, two-way ANOVA was performed, with Holm-Sidak's post hoc test comparing each 10 µm-section away from soma for both treatments. All data is presented as mean ± Standard Error Mean (SEM).

RESULTS

In order to check whether a single dose of 100 µg of 5-MeO-DMT increases cell proliferation in the adult DG as other serotonin 5-HT 1A agonists can, we labeled cells in S phase with BrdU. We found that 5-MeO-DMT treated animals showed a greater number of BrdU+ cells in the ventral DG compared to saline injected controls (saline treated: 155.4 ± 21.71 BrdU + cells per animal-see methods, n = 5 mice; 5-MeO-DMT treated: 352.6 ± 41.48 BrdU+ cells per animal, n = 5 mice. p = 0.0029, unpaired t-test, Figures). To investigate if the increase in cell proliferation was due to the recruitment of new progenitors or by an enhancement in progenitor division, we analyzed the cluster formation of the BrdU+ cells in DG in control and 5-MeO-DMT injected mice. Clustered cells suggest that they originate from the same progenitor, since newborn cells start to migrate right after proliferation. The total number of clusters in the ventral DG of 5-MeO- DMT treated animals was greater than saline (saline treated: 5.964 ± 0.5718 clusters per section, eight sections were analyzed per mice, n = 5 mice; 5-MeO-DMT treated: 11.7 ± 0.6974 clusters per section, eight sections were analyzed per mice, n = 5 mice. p = 0.0002, unpaired t-test, Figure). Moreover, we found a small difference in the number of cells per cluster between control and 5-MeO-DMT-treated mice trending to significance (saline treated: 1.433 ± 0.1365 cells per cluster, n = 5 mice; 5-MeO-DMT treated: 1.846 ± 0.119 cells per cluster, n = 5 mice. p = 0.0523, unpaired t-test, Figure). This data suggests that a greater number of progenitors are being recruited by 5-MeO-DMT. Following the proliferation assay, we tested if the number of newborn DG GC after ICV injection of 5-MeO-DMT is higher, aiming to answer if the 5-MeO-DMT also increase survivability of newborn neurons generated within the hippocampus. Our results indicate that the total number of DCX::tdTom+ cells is higher in the ventral hippocampus of 5-MeO-DMT treated mice (saline treated: 104 ± 6.348 DCX::tdTom+ cells, n = 6 mice; 5-MeO-DMT treated 220.3 ± 22.86 DCX::tdTom+ cells, n = 6 mice, p = 0.0006, unpaired t-test, Figures). Remembering that for the DCX-CreER T2 ::tdTom lox/lox mice, only a single hemisphere were used, not allowing accurate comparison between proliferation and survivability assays, however, the proportion of difference between the two treatments remain the same for both experimental frames. Next, we recorded if 5-MeO-DMT can modify electrophysiological properties of immature DG GC, we performed whole cell patch clamp onto DCX-CreER T2 ::tdTom lox/lox GC. In these experiments, DCX-CreER T2 ::tdTom lox/lox mice were perfused 21 days after ICV injections to study morphological differences in dendritic processes (Figure). Passive membrane and AP properties in response to a 500 ms-long 100 pA current step are shown in Table. Example membrane potential responses for a tdTomato+ cell from saline-and 5-MeO-DMT-injected mouse is shown in Figures. Cells from 5-MeO-DMT-treated animals exhibited higher AP threshold (Saline: -38.25 ± 2.03 mV n = 8 cells/3 animals; 5-MeO-DMT: -29.60 ± 2.51 mV, n = 11 cells/3 animals, p = 0.022, unpaired t-test, Figure). These cells also displayed a shorter AHP potential duration (Saline: 53.33 ms ± 12.04 ms n = 9 cells/3 mice; 5-MeO-DMT: 12.40 ms ± 1.302 ms, n = 8 cells/3 mice, p = 0.006, t-test, Figure) associated to a single fire pattern, five from eight saline treated cells displayed single fire pattern vs. none from 5-MeO-DMT treated group. AP threshold was defined as the voltage in which the rate of rise reaches a value superior to 20 mv/ms. We then applied current ramps (-50 pA to 200 pA in 1.5 s) in order to elucidate differences in fast activated currents between the two experimental groups. Example membrane potential responses to the current ramp is shown in Figure. Newborn GC from 5-MeO-DMT-treated mice showed a greater linear dependency between injected current and AP instantaneous frequency (Figures). The slope from the for the signal to go from 5% to 90% of its peak; * * * Latency defined as the time between the beginning of the current step protocol and the 5% of the first peak (all protocols have 100 ms delay); # Rate of Rise, defined as the maximum rate of change during the rising phase of the signal; ## AP threshold defined as the voltage point where the rate of rise reach a value superior to 20 mV/ms. AP half Width defined as the time taken to the signal rise from AP threshold to 50% its peak; AP after hyperpolarization (AHP) duration defined as the time beginning from 90% of AP peak until the voltage reach the value of 0% the peak again. linear regression ramp current (in pA) vs. AP instantaneous was equal to 0.11 ± 0.01 Hz/pA for controls and 0.17 ± 0.03 Hz/pA for 5-MeO-DMT group (n = 8 cells/three animals and n = 6 cells/three animals, respectively, p = 0.03, t-test, Figure). We have also patched randomly chosen tdTomato+ cells form DCX-CreER T2 ::tdTom lox/lox treated with saline (20 cells/four animals) or 5-MeO-DMT (16 cells/four animals) to test whether there was any cell in each group that did not fire in response to current injection. Three cells in the saline group (3/20 cells) did not fire APs while all cells fire APs in animals pre-treated with 5-MeO-DMT (p < 0.0001, z test). This data suggests that young GC from 5-MeO-DMT-injected mice show a higher degree of maturation than cells from control animals. We have then recorded sEPSCs from six mice (three in each group, 18 cells) treated or not with 5-MeO-DMT (Figure). While no difference in mean sEPSC half-width was found, sEPSC amplitude was greater in tdTomato+ cells of 5-MeO-DMTtreated mice im comparison to controls (57.15 ± 4.68 pA vs. 37.72 ± 6.60 pA, respectively, p = 0.03, t-test, Figure). Also, the frequency of sEPSC was drastically increased in tdTomato+ GC of 5-MeO-DMT-treated mice when compared to controls (1.35 ± 0.22 Hz vs. 0.35 ± 0.07 Hz, respectively, p = 0.002, t-test, Figure). These results indicate that newborn neurons from 5-MeO-DMT-treated mice are more prone to receive synaptic inputs from other DG cells. We then tested if 5-MeO-DMT also alters morphological maturation of newborn GC (tdTomato+ neurons, Figure). We first traced cells using an ImageJ plugin (see ''Materials and Methods,'' Section Figure) to later perform morphological analysis of dendrites. Number of branches in dendrite tree between treatment groups were different (saline treated: 7.15 ± 0.4247 dendritic branches, n = 20 cells from six mice; 5-MeO-DMT treated: 12.6 ± 0.916 dendritic branches, n = 15 cells from six mice, p = 0.0001, unpaired t-test, Figure). We then tested the same cells for dendritic complexity relative to cell nucleus, and the experimental group shows a higher number of intersections in the 50-170 µm range of distance from soma when compared to saline (saline intersect values: 50 µm, 3.4 ± 0.336; 60 µm, 3.4 ± 0.303; 70 µm, 3.850 ± 0.372; 80 µm, 3.750 ± 0.403; 90 µm, 3.650 ± 0.399; 100 µm, 3.6 ± 0.444; 110 µm, 3.350 ± 0.431; 120 µm, 3.350 ± 0.460; 130 µm, 2.950 ± 0.400; 140 µm, 2.5 ± 0.295; 150 µm, 2.2 ± 0.287; 160 µm, 1.850 ± 0.244; 170 µm, 1.55 ± 0.185, n = 20 cells from six mice; 5-MeO-DMT intersect values: 50 µm, 5 ± 0.406; 60 µm, 5.357 ± 0.464; 70 µm, 5.429 ± 0.456; 80 µm, 5.714 ± 0.450; 90 µm, 6.071 ± 0.559; 100 µm, 6.357 ± 0.561; 110 µm, 6.071 ± 0.606; 120 µm, 6.143 ± 0.653; 130 µm, 6.071 ± 0.752; 140 µm, 5.786 ± 0.735; 150 µm, 4.714 ± 0.699;

DISCUSSION

In this work we showed that a single dose of 5-MeO-DMT increases proliferation of neural progenitors and accelerates the maturation of newborn GC. We first used BrdU staining to show that 5-MeO-DMT treatment increases proliferation in the DG Next, we used an inducible Cre recombinase line under the control of a marker of neurogenesis (DCX) crossed with a fluorescent reporter to identify newborn neurons. In Figurewe show that the total number of DCX::tdTom+ cells in the ventral hippocampus of adult mice are increased, and this cells are likely DG GC, as was post hoc confirmed by electrophysiological, and morphological data that those cells are indeed neurons. Dendritic trees of newborn neurons from 5-MeO-DMT-treated mice were significantly more complex (with more branches and a higher number of intersections) when compared to saline-treated mice. AP threshold was lower and AHP potential was longer in newborn cells from 5-MeO-DMT-treated mice compared to controls. The higher number of BrDU+ cells indicate that a larger number of cells are entering in the S-phase of cell-cycle, but cannot elucidate the type of progenitor cell that is being affected. Studies using antibodies against GFAP, nestin and Sox2, might confirm if those BrdU+ cells are indeed RGL cells, the neural stem progenitors cells from adult DG. Also, future experiments may confirm whether the increase in BrdU+ cells following 5-MeO-DMT injection is due to the lengthening of S-phase or a higher recruitment of RGL (for example, using antibodies against ki67 or MCM2 associated with BrDU;. The choice of a single dose treatment, was made to address the gap between the molecular mechanisms, subjective and hormonal effects underlying Ayahuasca acute administration to depression diagnosed patients. The bulk of Ayahuasca tea, are composed of several psychoactive substances including DMT analogs and MAOi. The scope of present study is to unveil the effect of the 5-MeO-DMT, without adding any bias, due to other psychoactive compounds. To study the specific contribution of the 5-MeO-DMT to the adult neurogenic process, we needed to isolate the effect of the 5-MeO-DMT from other psychoactive components. In Ayahuasca tea the DMT is administrated with MAOi, in order to avoid tryptamines degradation. Using oral or intraperitoneal administration without MAOi may reduce the availability of 5-MeO-DMT to the central nervous system, since the monoamine oxidase will readily destroy any tryptamine, in the bloodstream, guts and also in the brain. Since 5-MeO-DMT can easily be degraded, we chose to deliver the 5-MeO-DMT i.c.v. to reduce the chemical inactivation prior to the arrival of the molecule to the brain. Additionally, it has been reported elsewhere that the harmine per se can increase neurogenesis, at least in vitro cultured hippocampal cells. Increased proliferation after 5-MeO-DMT injection does not indicate neuronal commitment. Thus, we performed histological analysis in DCX-CreER T2 ::tdTom lox/lox mice injected with 5-MeO-DMT. Our results indicate a greater number of DCX::tdTom+ cells in the ventral hippocampus of 5-MeO-DMT treated animals, showing that the total numbers of neuron that reach neuronal maturity are also increased, in addition to the initial increase in proliferation right after 5-MeO-DMT injection as evinced by our proliferation assay Figure. Serotonin has been shown to increase granule cell proliferation in the adult DG. However, serotonin does not seem to affect specialization of newborn cells in the SGZ. Our results, on the other hand, suggest that 5-MeO-DMT not only has a positive effect on proliferation and survivability, but also on the maturation of GC. Hence, our results imply that the positive effect of 5-MeO-DMT in adult neurogenesis differs from that of serotonin alone. Our current-clamp recordings indicate that young neurons from 5-MeO-DMT-treated mice show faster maturation than cells from control animals. Mature GC show a higher AP threshold and are able to fire in higher frequencies). These differences in maturation were also found in the morphology of dendritic trees. Dendritic complexity is a major indicative of cell maturation. Cells from animals submitted to a single 5-MeO-DMT injection showed dendrites with more branches and intersections. Interestingly, chronic antidepressant therapy also accelerates the maturation of dendrites. Future studies should address how tryptamine analogs affect the temporal expression of voltage-dependent currents. Our preliminary results indicate, for example, that the hyperpolarizing-activated current, I h , is larger in novel GC in animals injected with 5-MeO-DMT when compared with saline Also, it will be interesting to examine changes in Cl -reversal potential as GC show a depolarized potential until adolescence. Dorsal Raphe Nucleus profusely targets the SGZbut a previous work have shown that lowering serotonin levels in the brain can increase neurogenesis). Yet, serotonin agonists and serotonin uptake inhibitors seem to increase neurogenesis. Hence, specific 5HT receptors might be involved in neurogenesis modulation. 5-HT 1A , 5-HT 2A and 5-HT 2C , 5-MeO-DMT targets, are all expressed in the DG (Allen Institute for Brain Science 1 , experiments n • : 79394355, 81671344 and 71393424, respectively). While 5-MeO-DMT is a strong 5-HT 2A and 5-HT 2C agonist, this compound acts in other receptors (with much lower potency). Hence, we cannot affirm that the effect of 5-MeO-DMT in neurogenesis occurs through 5-HT 2A and 5-HT 2C receptors. Future studies using agonists and antagonists are necessary for dissecting the molecular mechanism of 5-MeO-DMT action in neurogenesis. In conclusion, we show here that a single dose of 5-MeO-DMT can increase proliferation, survivability and accelerate maturation of newborn neurons in the DG. To our knowledge, this work was the first to demonstrate a direct effect of a naturally occurring psychoactive compound in adult neurogenesis. New lines of investigation have suggested that serotoninergic hallucinogens can significantly improve severe depression and anxiety. Thus, the effect of 5-MeO-DMT in modulating neurogenesis could throw light on the mechanism behind the beneficial effects of hallucinogenic compounds in mood disorders.