One of the main natural components of ayahuasca tea is dimethyltryptamine (DMT), which promotes neurogenesis – the formation of new neurons – according to research led by the Complutense University of Madrid (UCM).
In addition to neurons, the infusion used for shamanic purposes also induces the formation of other neural cells such as astrocytes and oligodendrocytes.
“This capacity to modulate brain plasticity suggests that it has great therapeutic potential for a wide range of psychiatric and neurological disorders, including neurodegenerative diseases,” explained José Ángel Morales, a researcher in the UCM and CIBERNED Department of Cellular Biology.
The study, published in Translational Psychiatry, a Nature Research journal, reports the results of four years of in vitro and in vivo experimentation on mice, demonstrating that these exhibit “a greater cognitive capacity when treated with this substance,” according to José Antonio López, a researcher in the Faculty of Psychology at the UCM and co-author of the study.
Changing the receptor eliminates the hallucinogenic effect
Ayahuasca is produced by mixing two plants from the Amazon: the ayahuasca vine (Banisteriopsis caapi) and the chacruna shrub (Psychotria viridis).
The DMT in ayahuasca tea binds to a type-2A serotonergic brain receptor, which enhances its hallucinogenic effect. In this study, the receptor was changed to a sigma type receptor that does not have this effect, thus “greatly facilitating its future administration to patients.”
In neurodegenerative diseases, it is the death of certain types of neurons that causes the symptoms of pathologies such as Alzheimer’s and Parkinson’s. Although humans have the capacity to generate new neuronal cells, this depends on several factors and is not always possible.
“The challenge is to activate our dormant capacity to form neurons and thus replace the neurons that die as a result of the disease. This study shows that DMT is capable of activating neural stem cells and forming new neurons,” concluded Morales.
N,N-dimethyltryptamine (DMT) is a natural compound found in numerous plant species and botanical preparations, such as the hallucinogenic infusion known as ayahuasca1 classified as a hallucinogenic compound that induces intense modifications in perception, emotion, and cognition in humans2–4.
DMT is present in several animal tissues, such as the lung5 and brain6, being considered as an endogenous trace neurotransmitter with different physiological roles, including neural signaling and brain/peripheral immunological actions7–10. DMT is also present in human blood, urine, and cerebrospinal fluid11–13.
Furthermore, some evidence suggests that DMT can be sequestered into and stored in the vesicle system of the brain and that environmental stress increases its levels in mammals’ central nervous system (CNS)14–16. DMT binds and exerts an agonist activity on subtypes 1A and 2A of the serotonin receptor (5-HT)17,18. T
hese receptors are G-protein-coupled receptors (GPCRs) belonging to the family of serotonergic receptors and are involved in numerous cascades of intracellular signaling, with high expression in several regions of the CNS. Some studies have demonstrated that DMT also binds with low affinity to non-serotonergic receptors, such as the sigma-1 receptor (S1R).
The S1R, traditionally thought to be an opioid receptor, is now classified as a highly conserved transmembrane protein member of an orphan family and located mainly in the membrane of the endoplasmic reticulum. σR‐1 is widespread in the CNS, mainly in the prefrontal cortex, hippocampus, and striatum19. Interestingly, in mammals, one of the natural endogenous ligands of the σR-1 is DMT14.
This receptor has been associated with several cellular functions, including the brain, such as lipid transport, metabolism regulation, cellular differentiation, signaling (in response to stress), cellular protection against oxidant agents, myelination and, most recently, neurogenesis20–24.
Neurogenesis is the process of generating new functional neurons, mainly in the SVZ and the subgranular zone of the DG of the hippocampus. In mammals, this process occurs mostly during the prenatal period, being significantly reduced in adults25–30. In humans, although the presence of adult neurogenesis has been recently reported during aging31–33, most of the studies indicate that there are no substantial evidence to support it.
A recent review by Duque and Spector suggests that, in adult age, preservation of the existing neurons is more important in contrast to the generation of new ones34. Neurogenesis is a complex process involving multiple cellular activities including the proliferation of neural stem cells (NSC; progenitors), migration and differentiation, survival, acquisition of cell fate and maturation, and integration of these newly born neurons in existing neuronal circuits.
All these processes are precisely regulated by multiple factors35. Advancements in the knowledge of these factors and their mechanism of action could help us to investigate possible new instruments that will allow us to expand the limited endogenous neurogenic capacity of the adult brain and, consequently, opening new fields for the development of effective therapies in the treatment of brain damage and neurodegenerative diseases.
Neurodegenerative diseases (including Parkinson’s, Alzheimer’s, Hungtinton’s, etc.) and acute neural damage (such as stroke and traumatic brain injury) are characterized by a gradual and selective loss of neurons in the affected regions of the nervous system.
One common feature in these disorders is an impairment in the proliferation of progenitor cells in the neurogenic niches36,37. In animal models reproducing the pathological hallmarks of Alzheimer disease, a loss of neurogenic capacity has been described in the SVZ38.
This decrease is also observed in the postmortem brains of Parkinson’s patients, suggesting that the loss of neurogenic activity is due to the loss of dopamine, affecting the neural precursors in the adult39. These data support the fact that in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, not only degeneration and death of mature neurons occur but also the process of formation of new neuronal progenitors in the adult brain is negatively affected.
According to these data, the stimulation of endogenous populations of stem cells and neuronal progenitors could be a promising approach to improve the functionality of some of the regions affected by neurodegenerative pathologies. In fact, the stimulation of neurogenesis has already been proposed as a new therapeutic strategy for psychiatric and neurological diseases40–44, and several studies have reported that the clinical efficacy of antidepressant drugs is frequently linked to the capacity of these drugs to induce neurogenesis45–48.
Based on the data above mentioned including our results on the potent neurogenic effect of the other components of the Ayahuasca49, the main objective of this work was to analyze the possible role of DMT in adult neurogenesis, as well as to elucidate its mechanism of action.
Results
DMT controls the stemness of neural progenitors in vitro through the S1R
We first analyzed whether the sigma-1 receptor (S1R) was expressed on murine NSCs isolated from the subgranular zone of the dentate gyrus of the hippocampus. Figure 1a shows S1R expression on neurospheres in the basal state determined by immunocytochemistry and western blot analysis.
To analyze the “stemness” of cultured neurospheres, we determined the expression of potentiality markers of this state. Then, we performed WB analysis after treatment of these cultures during 7 days under proliferative conditions (see “Materials and methods”) with DMT alone or in combination with the different antagonists. Our results (Fig. 1b) show significant reductions in protein levels of musashi-1, nestin, and SOX-2 in the SGZ-derived neurospheres after treatment with DMT, suggesting a loss of stemness in NSCs in the NS cultures.
When these cultures were pre-treated with BD1063, a specific antagonist for S1R, this effect was reversed, and stemness marker levels were similar to those observed in basal conditions. On the contrary, the expression of stemness markers in those cultures treated with DMT together with the mixed serotonin 5-HT1A/2A receptor antagonist methiothepin, the selective 5-HT2A receptor antagonist ritanserin, or the selective 5-HT1A receptor antagonist WAY100635, significantly decreased stemness as occurred in DMT-treated cultures. These results suggest that DMT promotes a loss of “stemness” or and undifferentiated state of the neurospheres, through the S1R.
DMT promotes the proliferation in vitro of NSCs
Other NS cultures were used to study proliferation; thereby, the number and diameter of the neurospheres were evaluated (Fig. (Fig.1c).1c). DMT notably increased the number and size of the neurospheres in NS cultures after 7 days of treatment, indicating that DMT promotes the proliferation of adult hippocampal-derived neural progenitors.
DMT proliferative effect was blocked when cultures were treated with BD1063 showing a significant decrease in the number and size of neurospheres, similar to basal conditions. Also, significant differences, in number and size of neurospheres, were observed when cultures were treated with DMT combined with methiothepin, ritanserin, or WAY100635.
We next analyzed changes in two well-known markers for proliferation, ki67 and proliferating cell nuclear antigen (PCNA) (Fig. 1d, e). Fluorescent immunocytochemical analysis of ki67 expression (Fig. (Fig.1d)1d) showed an increase in the number of ki67+ cells in the NS after treatment with DMT, suggesting a direct effect of DMT on the proliferation ability of NSCs. This effect was clearly reverted when cultures were also incubated with the antagonist BD1063 (BD).
Similar results were obtained by western blot analysis and subsequent quantification of PCNA (Fig. (Fig.1e).1e). No significant differences in the expression of ki67 and PCNA were observed when cultures were preincubated with other DMT antagonists. These results indicate that DMT stimulates in vitro, through the S1R, the proliferation of neural progenitors of the adult neurogenic niche of the hippocampus.
DMT promotes the differentiation in vitro of NSCs toward the three main neural cellular types
Treated neurospheres during 7 days in the presence of DMT, alone or in combination with the different antagonists, under differentiation conditions (medium with 1% fetal bovine serum and absence of growth factors) were used. To study the ability to differentiate into a certain neural phenotype, the expression of specific proteins linked to every neural subtype was analyzed (Fig. (Fig 2). To detect neurons, β-III-tubulin (clone TuJ-1), found exclusively in neurons and MAP-2 (microtubule-associated protein 2), present in mature neurons, were used (Fig. 2a, b). To study its differentiation toward an astroglial or oligodendroglial phenotype (Fig. 2c, d), we analyzed the expression of GFAP (astrocytes) and CNPase (oligodendrocytes).
N,N-dimethyltryptamine (DMT) promotes stem cell differentiation toward all neural phenotypes.
SGZ-derived neurospheres were cultured for 7 days in the presence of DMT alone or in combination with the antagonists BD1063 (BD), methiothepin (Met), ritanserin (Rit), and WAY100635 (WAY) and then were adhered on a substrate and allowed to differentiate for 3 days. a Confocal fluorescent images showing the expression of the neuronal markers β-III-Tubulin (TuJ-1 clone, green) and MAP-2 (red) in NS (n = 8 per condition). DAPI was used for nuclear staining. Scale bar = 50 μm. b Representative western blots and quantification of β-tubulin and MAP-2. c Immunofluorescence images showing NS expressing the glial fibrillary acidic protein (GFAP, red) that stains astrocytes, and in green, the oligodendrocyte marker CNPase (n = 8 per condition). DAPI was used for nuclear staining. Scale bar = 50 μm. d Representative western blots of CNPase and GFAP and quantification. Results are the mean ± SD of four different cellular pools with three independent experiments/pool (n = 12). After confirming the significance of the primary findings using ANOVA, a significance level of P < 0.05 was applied to all remaining post hoc (Tukey test) statistical analyses. **P ≤ 0.01; ***P ≤ 0.001 indicate significant results versus non-treated (basal) cultures.
Figure 2a, b shows a striking increase in the expression of β-III-tubulin and MAP-2 in neurospheres treated with DMT, compared with basal (non-treated) cultures. This neurogenic effect is clearly blocked by BD. No differences in the expression of neuronal markers were observed when cultures were treated with DMT in combination with metiotepine, ritanserin, or WAY. These results suggest that DMT stimulates the in vitro differentiation of neural progenitors toward a neuronal phenotype through S1R.
Related to gliogenesis, Fig. 2c, d shows an increase in the expression levels of GFAP and CNPase, after DMT treatment. This promotion of astroglial cells and oligodendrocytes generation was blocked when cultures were pre-treated with the antagonist BD. We did not observe differences in the expression of GFAP and CNPase when neurospheres were pre-treated with the other antagonists. These results may suggest a direct effect of DMT on in vitro differentiation of neural progenitors toward astrocytes and oligodendrocytes via S1R.
DMT activates in vivo the subgranular zone neurogenic niche in adult mice
To confirm our in vitro results on the role of the S1R on DMT neurogenic action, we first determined the expression of S1R in the subgranular neurogenic niche. To that end, brain coronal sections, including the hippocampus and protein samples isolated from the SGZ, were analyzed. As can be observed in Fig. 3a, immunofluorescence on the subgranular zone and western blot analysis shows the expression of S1R in this brain area. We next analyzed whether DMT also exerted an effect stimulating the proliferation kinetics of NSCs in the SGZ in vivo.
To that end, adult mice were intraperitoneally injected during 4 (short-term) or 21 days (long-term) with DMT alone or in combination with antagonists, followed by BrdU administration for 24 h (Fig. 3) or 21 days (Fig. 4) before sacrifice. In short-term animals (Fig. 3b), immunohistochemical and cell count analysis performed on brain serial coronal sections containing the SGZ (Fig. 3c, d) demonstrated that DMT significantly increased the number of double BrdU/Nestin-stained cells in the SGZ, in comparison with control values (clorgyline-treated group).
This neurogenic stimulation seemed to be mediated by the S1R since no neurogenic effect was observed when DMT is administered together with the antagonist BD1063. No differences were found in BrdU and nestin immunostaining in those animals injected with DMT in combination with the antagonist methiothepin and WAY100635.
N,N-dimethyltryptamine (DMT) promotes in vivo activation of the neurogenic niche located in the SGZ of the dentate gyrus in the hippocampus.a Representative images (n = 3 animals) showing the expression of the sigma-1 receptor (S1R, green) on neural stem cells (NSCs) (red-labeled with nestin) in the subgranular zone of the dentate gyrus. S1R expression was also determined by western blot on hippocampal tissue (n = 3 animals). b Schematic representation of experimental design and treatment schedule for in vivo short-term neurogenic study. DMT alone or in combination with the antagonists BD1063 (BD), methiothepin (Met), ritanserin (Rit), or WAY100635 (WAY) was daily intraperitoneally injected during 4 days. To label proliferating cells, on day 4 mice were i.p. injected with 5-bromo-2-deoxyuridine (BrdU) and sacrificed on day 5. c Confocal maximum intensity projection images showing BrdU/Nestin co-staining on the SGZ of adult mice. BrdU is shown in green and nestin in red. Scale bar = 25 μm. d Quantification of the number of double BrdU/Nestin cells in the DG performed on confocal orthogonal images is shown. e Double BrdU+-DCX+ expressing cells in the SGZ. Confocal maximum intensity projection images show BrdU in green and DCX in red. Scale bar = 25 μm. f Quantification of the number of BrdU+– DCX+ cells in the DG is shown. All quantification values represent the mean ± SD (n = 5 animals per group). After confirming the significance of the primary findings using ANOVA, a significance level of P < 0.05 was applied to all remaining post hoc (Tukey test) statistical analyses. **P ≤ 0.01; ***P ≤ 0.001 indicate significant results versus vehicle-treated (basal) animals.
N,N-dimethyltryptamine (DMT) promotes in vivo neurogenesis on the subgranular zone of the dentate gyrus in the hippocampus.
a Schematic representation of the experimental design and treatment schedule for in vivo long-term neurogenic study. DMT alone or in combination with the antagonists BD1063 (BD), methiothepin (Met), ritanserin (Rit), or WAY100635 (WAY) was intraperitoneal injected on alternate days during 21 days. To label proliferating cells, on day 1 mice were i.p. injected with 5-bromo-2-deoxyuridine (BrdU) and sacrificed on day 21. b BrdU-DCX-expressing cells in the DG. Representative confocal images are shown. Scale bar = 25 μm. c Quantification of the number of BrdU+-DCX+-expressing cells in the DG based on confocal orthogonal images. d Confocal maximum intensity projection images showing the colocalization of BrdU (green) and neuN (red) cells in the dentate gyrus of the hippocampus of adult mice. Scale bar = 25 μm. e Quantification of the number of BrdU+/NeuN+ cells performed on confocal orthogonal images in DG. Values represent the mean ± SD (n = 5 animals per group). After confirming the significance of the primary findings using ANOVA, a significance level of P < 0.05 was applied to all remaining post hoc (Tukey test) statistical analyses. **P ≤ 0.01; ***P ≤ 0.001 indicate significant results versus vehicle-treated (basal) animals.
During the neurogenic process, proliferation is crucial but also the migration of the newly generated precursor from the SGZ to the granular layer. To study the migration of neural precursors, serial coronal brain sections were stained for doublecortin (DCX).
The results shown in Fig. 3e, f show a higher immunopositive BrdU/DCX cells in the SGZ of DMT-treated animals. ùAdditionally, DCX-stained cells in DMT-treated animals exhibited extensive dendritic arborizations. No difference was observed when animals were treated with DMT combined with antagonists.
Contrarily, when DMT was injected with BD1063, Brdu, or DCX expression was not increased. These results confirm that DMT-treated mice exhibit enhanced proliferation and migration of neural precursors in the SGZ after 4 days of treatment, suggesting a modulating effect of this compound on hippocampal neurogenesis in vivo.
In order to know whether these new migrating neuroblasts were able to properly reach the granular cell layer, long-term (21 days) treated animals were used (Fig. 4a). Quantification analysis of confocal images demonstrates an increase in DCX+/BrdU+-cell in the SGZ after DMT treatment (Fig. 4b, c). No differences were found in those animals treated with DMT together with methiothepin or WAY100635. Once again, combined treatment of DMT with BD1063 blocked the migration increase observed in animals treated only with DMT.
In addition, at this time when neuroblasts have reached the granular cell layer, a noticeable increase in the amount of newly generated neurons (BrdU+/NeuN+ cells) was seen in this layer (Fig. 4d, e) in DMT-treated animals. This increase in the number of newly generated granular cells was blocked when mice treated with DMT together with BD1063. Altogether, these observations clearly indicate that DMT increases in vivo the number of new neurons originated in the hippocampus, action mediated by S1R.
Taking into account these results, we finally analyzed the functional consequences of DMT treatment by performing behavioral tasks (Fig. (Fig.5a)5a) to analyze if memory and learning are affected. Figure Figure5b5b (left panel) shows the results obtained by the Morris water maze test. During the learning curve, there were significant differences between groups only on days 4 and 5, showing that the DMT group tended to reduce the escape latency compared with DMT + ritanserin and control groups, respectively. In the probe trial, DMT and DMT + ritanserin groups showed a significant reduction in escape latency compared with the control group.
In this line, we found that the control group performed less platform crosses and spent less time in target annulus around the previous platform location. ùData from the probe trial indicate that the DMT and DMT + ritanserin remembered more effectively the zone where the hidden escape platform was.
During the 3 days of cued learning, no differences were observed between groups, indicating that the differences observed in the learning curve and probe trial were not due to differences in the motivation of animals to escape from the water nor sensorimotor abilities.
Regarding the new object recognition test (Fig. 5c, right panel), the DMT group showed a longer exploration time of the new object and a greater number of approaches to it. In addition, this group tended to explore the new object before the old one. DMT + ritanserin group spent more time exploring the new object and approached it more times. Finally, the control group only spent more time exploring the new object.
In addition, we obtained differences in the latency of the first approach and exploration time between DMT and control groups. These results suggest that DMT and DMT + ritanserin groups showed better episodic memory compared to the control group.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522265/
More information: Jose A. Morales-Garcia et al, N,N-dimethyltryptamine compound found in the hallucinogenic tea ayahuasca, regulates adult neurogenesis in vitro and in vivo, Translational Psychiatry (2020). DOI: 10.1038/s41398-020-01011-0
