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

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.

Figure 1

Effects of ayahuasca β-carboline alkaloids on stemness of cultured adult neurospheres. Representative Western blots and bar graphs showing expression levels of the precursor cell markers musashi-1, nestin and SOX-2 after treatment with each of the four alkaloids tested (1 µM). Values in bar graphs indicate mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for the subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **P ≤ 0.01; ***p ≤ 0.001 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.

Figure 2

Effects of ayahuasca β-carboline alkaloids on adult neurosphere formation. (a) Representative phase-contrast micrographs showing the number and size of neurospheres after 7 days in culture in the presence of each of the four alkaloids tested (1 µM). The number and diameter of at least 50 neurospheres was determined in control and treated cultures. Scale bar = 100 μm. (b) Bar graphs showing results as mean values ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for he subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.

Figure 3

Effects of ayahuasca β-carboline alkaloids on adult neural stem cells proliferation. (a) Representative immunofluorescence images showing the expression of the cellular marker for proliferation ki67 (green) in neurospheres derived from the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. SVZ-derived neurospheres are shown in two panels showing the central part of the sphere (left) and the distal migration site (right). Single images from SGZ-derived neurospheres show the whole neurosphere, including the central and distal areas. DAPI was used for nuclear staining. Scale bar = 50 μm. (b) Representative Western blots of ki67 and the proliferating cell nuclear antigen (PCNA) levels in neurospheres treated for 7 days with each of the four alkaloids tested (1 µM). Bar graphs show the results of the quantification analyses. Each bar indicates relative protein levels expressed as mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for he subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **p ≤ 0.01 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.

Figure 4

Ayahuasca β-carboline alkaloids promote stem cell differentiation towards a neuronal phenotype. After 7 days on culture in the presence of harmol, harmine, harmaline and tetrahydroharmine (THH), free floating neurospheres derived from the adult subgranular (SGZ) and subventricular (SVZ) zone were adhered on coated coverslips and allowed to differentiate for 3 days in the presence of alkaloids at 1 µM. (a) Representative immunofluorescence images showing the expression of the neuronal markers β-III-Tubulin (TuJ-1 clone, green) and MAP-2 (red) in neurospheres. DAPI was used for nuclear staining. SVZ-derived neurospheres are shown in two panels showing the central part of the sphere (left) and the distal migration site (right). Single images from SGZ-derived neurospheres show the whole neurosphere, including the central and distal areas. Scale bar = 50 μm. (b) Representative Western blots of β-tubulin and MAP-2. Quantification analyses are also shown. Results are the mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for he subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **p ≤ 0.01 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.

Figure 5

Ayahuasca β-carboline alkaloids promote astrogliogenesis. Neurospheres derived from the adult subgranular (SGZ) and subventricular (SVZ) zone were cultured in the presence of harmol, harmine, harmaline and tetrahydroharmine (THH). After 7 days neurospheres were adhered on coated coverslips and allowed to differentiate for 3 days in the presence of alkaloids at 1 µM. (a) Neurosphere immunofluorescence images showing in green the expression of CNPase (oligodendrocyte marker) and glial fibrillary acidic protein (GFAP, red) that stains astrocytes. SVZ-derived neurospheres are shown in two panels showing the central part of the neurosphere (left) and the distal migration site (right). Single images from SGZ-derived neurospheres show the distal part of the neurosphere. DAPI was used for nuclear staining. Scale bar = 50 μm. (b) Representative Western blots of CNPase and GFAP. Quantification analyses are also shown. Results are the mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for he subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **p ≤ 0.01 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.