The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro

Regarding the reportedly anti-depressant effect of ayahuasca, this study (2017) found that the three main alkaloids contained in B. caapi (one of the two main ingredients of ayahuasca) stimulate adult neurogenesis/plasticity in vitro (new neuronal cells in a dish).

Abstract

“Banisteriopsis caapi is the basic ingredient of ayahuasca, a psychotropic plant tea used in the Amazon for ritual and medicinal purposes, and by interested individuals worldwide. Animal studies and recent clinical research suggests that B. caapi preparations show antidepressant activity, a therapeutic effect that has been linked to hippocampal neurogenesis. Here we report that harmine, tetrahydroharmine and harmaline, the three main alkaloids present in B. caapi, and the harmine metabolite harmol, stimulate adult neurogenesis in vitro. In neurospheres prepared from progenitor cells obtained from the subventricular and the subgranular zones of adult mice brains, all compounds stimulated neural stem cell proliferation, migration, and differentiation into adult neurons. These findings suggest that modulation of brain plasticity could be a major contribution to the antidepressant effects of ayahuasca. They also expand the potential application of B. caapi alkaloids to other brain disorders that may benefit from stimulation of endogenous neural precursor niches.”

Authors: Jose A. Morales-García, Mario de la Fuente Revenga, Sandra Alonso-Gil, María Isabel Rodríguez-Franco, Amanda Feilding, Ana Perez-Castillo & Jordi Riba

Summary

Ayahuasca is the Quechua name for Banisteriopsis caapi, a jungle liana of the Malpighiaceae family. It is used in shamanic practices and rites of passage by the indigenous inhabitants of northwestern South America.

Ayahuasca contains high amounts of harmine and tetrahydroharmine, three indole alkaloids with -carboline structure, and can inhibit monoamine-oxidase-A (MAO-A) and serotonin reuptake. It can also contain up to 100 different plants.

In recent years, non-traditional use of ayahuasca has expanded worldwide, and studies have found that a single dose of ayahuasca rapidly reduced depressive symptoms in treatment-resistant patients. However, the -carbolines of B. caapi may play a minor role in the overall pharmacology of ayahuasca preparations.

The -carbolines present in ayahuasca may contribute to the CNS effects of the tea, which have been observed in animal models of depression. Additionally, harmine increases BDNF levels in the hippocampus after both acute and chronic administration.

Antidepressants stimulate adult neurogenesis in two main niches: the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus of the hippocampus. These newly generated neurons have the capacity to migrate and integrate into existing neural circuits.

Here, we investigated the capacity of the three main -carbolines present in B. caapi to induce neurogenesis in vitro using neural progenitor cells from adult mice. The results showed that the alkaloids directly regulate proliferation, migration and differentiation of neural stem cells.

We isolated neural stem cells from the SVZ and SGZ of B. caapi and cultured them as free-floating neurospheres. Treatment with the four -carbolines reduced the amount of musashi-1, nestin and SOX-2 in the neurospheres, indicating a loss of “stemness” or undifferentiated state of the neurospheres.

B. caapi -carbolines increased the rate of formation and size of neurospheres in cultures with epidermal growth factor and fibroblast growth factor.

Results

After 7 days of treatment with ayahuasca -carboline alkaloids, the number of SVZ-derived neurospheres was significantly higher than in the vehicle-treated cultures, and the size of the neurospheres was significantly increased as compared with non-treated cultures.

Figure 2 shows the effects of ayahuasca -carboline alkaloids on adult neurosphere formation. The results are shown as mean values SD for at least three independent experiments corresponding to four different cellular pools.

Ayahuasca-carboline alkaloids stimulated the proliferation of adult neural stem cells in the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampus. The results indicated that the alkaloids had a significant effect on the proliferation of these cells.

The treatment of cultures with the -carbolines resulted in an increase in the number of Ki67- and PCNA-positive cells, indicating a direct effect of these compounds on proliferation.

B. caapi -carbolines increased neural stem cell migration in neurospheres, and the cells moved out of the neurosphere body in the presence of the compounds, whereas the cells in the control cultures remained close to the neurosphere core.

B. caapi -carbolines induce differentiation of neural stem cells into astrocytes, neurons and/or oligondendrocytes. The test compounds increased the number of Tuj1- and MAP-2-positive cells in the cultured cells, indicating that they induce the differentiation of neural stem cells towards mature neurons.

The results show that most of the test compounds did not induce the differentiation of neural precursors towards an oligodendrocyte phenotype. However, harmol- and harmaline-treated SVZ cultures presented some scattered CNPase-positive cells and GFAP-positive cells.

Discussion

Adult neural stem cell activity is regulated by harmine, THH, and harmaline, the most abundant alkaloids in B. caapi and ayahuasca, and by harmol, the main metabolite of harmine in humans31. These alkaloids promote the proliferation and migration of progenitor cells and promote their differentiation predominantly into neurons.

The effects of harmine and THH on neurogenesis have not been studied before, but harmine increases the number and size of neurospheres, the number of Ki-67-stained cells, and the amounts of Ki-67 and PCNA protein as measured by Western blot.

Our results showed that B. caapi -carbolines promoted cellular migration and differentiation. The largest effects on migration were observed for harmaline and THH, and all tested compounds promoted cellular differentiation into neurons, astrocytes, and oligodendrocytes.

Ayahuasca -carboline alkaloids promote stem cell differentiation towards a neuronal phenotype. This was demonstrated by the expression of the neuronal markers -III-Tubulin (TuJ-1 clone, green) and MAP-2 (red) in neurospheres derived from the adult subgranular zone (SGZ) and subventricular zone (SVZ) of the brain.

Ayahuasca -carboline alkaloids promote astrogliogenesis in neurospheres derived from the adult subgranular (SGZ) and subventricular (SVZ) zone of the hippocampus. CNPase and GFAP are expressed in the oligodendrocytes and astrocytes, respectively, in the subgranular zone and the subventricular zone, respectively.

All of the above indicate that the B. caapi -carbolines stimulate neurogenesis at multiple levels, and that they can act simultaneously on various processes.

B. caapi -carbolines increased monoamine levels, which could explain the observed effects of -carbolines in neurogenesis. However, the role of monoamines in neurogenesis is not fully understood, and future studies should assess whether -carbolines interact with one or more of the following pathways.

Our findings have relevant therapeutic implications. The association between neurogenesis and anti-depressant activity is well documented, and clinically effective antidepressants stimulate neurogenesis, independent of their chemical structure and mechanism of action.

Ayahuasca has been shown to have antidepressant effects in humans and may have potential anti-addictive properties. It has also been shown to have neurogenic niche stimulation properties, which may be a novel therapeutic strategy for neuropsychiatric disorders.

Our study has a series of limitations, including the fact that ayahuasca brews contain other active compounds that were not tested here. However, future studies could test the four compounds assessed here for both in vivo neurogenesis and behavioral improvement of depression.

Ayahuasca tea contains -carboline alkaloids, which stimulate neural progenitor pool expansion and induce cellular migration and differentiation into a neuronal phenotype. This may contribute to the antidepressant effects of ayahuasca.

Methods

1-Methyl-9H-pyrido[3,4-b]indol-7-ol hydrobromide (harmolHBr) was synthesized from harmine by heating the mixture at 160 °C during 25 min in a microwave oven and then allowed to cool to room temperature. The product was purified by HPLC and characterized by NMR and HRMS.

7-Methoxy-1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole hydrochloride was synthesized from harmaline by adding sodium borohydride to a stirred solution of harmaline in water at 0 °C. The product was collected by filtration and identified by 1H NMR, 13C NMR and HPLC.

Adult precursors were isolated from the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle of C57BL/6 mice and seeded into 12-well dishes containing 10 ng/mL epidermal growth factor and 10 ng/mL fibroblast growth factor.

Neurospheres were cultured in 12-well dishes with growth factors and treated daily with 1 M solutions of each of the four -carbolines tested: harmol, harmine, harmaline and tetrahydroharmine (THH). At these concentrations, none of the tested -carbolines affected the viability of the cultured cells. Proliferation and growth of neurospheres were assessed using Nikon Digital Sight, SD-L1 software, and some were used for immunoblotting analysis. Others were differentiated onto poly-L-lysine precoated-6-well plates and/or coverslips and used for migration assays.

Immunocytochemistry was performed on NS grown on glass. Ki67, -III-tubulin, MAP-2, CNPase and GFAP were detected and stained with Alexa-488 and Alexa-647 antibodies, and images were acquired using a LSM710 laser scanning spectral confocal microscope.

Differentiated cultured NS were lysed with protease inhibitor cocktail, loaded on a 10% or 12% SDS-PAGE gel, blocked with Tris-buffered saline with 0.05% Tween-20 and 5% skimmed milk or 4% BSA, incubated with primary and secondary antibodies, and washed according to standard procedures.

Cellular migration assay was performed using a Cell Observer system from Zeiss, equipped with a Cascade 1 K camera, a monitorized X/Y stage, and a Module S incubator with equipment for temperature and CO2 control. Phase contrast images were taken every 60 min using a 4 objective.

Statistical analysis was performed using a one-way ANOVA with treatment as factor. A pair-wise test was performed on significant results.

Acknowledgements

This work was supported by the MINECO, FEDER and the Beckley Foundation. J.A.M-G. is a post-doctoral fellow at CIBERNED.

Author Contributions

A.P.-C., J.R., M.F.-R., S.A.G., M.I.R.-F. and A.F. participated in the project, designed the experiments, analyzed the data and wrote the manuscript.

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