Here we showed that 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. Using an in vitro model of adult neurogenesis, we found that all four β-carbolines stimulated the proliferation and migration of progenitor cells and promoted their differentiation predominantly into neurons.

The four compounds tested effectively promoted proliferation, migration, and differentiation of progenitor cells obtained from the SVZ and the SGZ, the two main niches of adult neurogenesis in rodents. The β-carbolines increased the number and size of primary neurospheres, induced the loss of the neurospheres’ undifferentiated state, and promoted subsequent cell migration and differentiation mainly into a neuronal phenotype, as indicated by the positive expression of β-III-tubulin and MAP2, but also into astroglial cells. Taken together, these three effects indicate that B. caapi alkaloids have the capacity to regulate the expansion and fate of stem cell populations.

Analysis of the proliferation stage showed that all four β-carbolines increased 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 for the effect of harmine on proliferation are in line with a previous study showing harmine-induced increase in mitosis in cultured chick embryo cells32, and in human neural progenitor cells33. To our knowledge, the effects of harmaline and THH on neurogenesis have not been studied before. While harmaline is only present in small amounts in B. caapi, THH is the second most abundant β-carboline in the plant4, 29. Additionally, THH shows more consistent plasma levels between individuals and studies than harmine, which is rapidly degraded to harmol when taken orally29, 34. The latter, formed in vivo by O-demethylation of the parent compound29, showed proliferative effects of similar magnitude to those of harmine.

Our results showed that B. caapi β-carbolines promoted cellular migration and differentiation, suggesting that these alkaloids not only act as mitogens for neural stem cells, but also modulate cellular fate. The largest effects on migration were observed for harmaline and THH. Increased migration capacity is relevant in certain conditions such as brain injury, where stem cell niches are far from the damaged area35,36,37. All tested compounds also promoted cellular differentiation. Neural stem cells are known to differentiate into neurons, astrocytes, and oligodendrocytes23, 24. The observed increases in Tuj-1 and MAP-2 protein expression indicated differentiation predominantly toward a neuronal phenotype. In the SVZ both proteins were equally expressed after each of the four treatments. However, in the SGZ harmine administration did not influence Tuj-1 levels, a marker of immature neurons, but significantly increased the expression of MAP-2, suggesting a larger impact on neuronal maturation.

All the above indicate that B. caapi β-carbolines facilitate neurogenesis at multiple levels. This capacity is of interest, since in pathological conditions the replacement of neurons may be optimized by acting simultaneously on various processes38, 39. The effect of the β-carbolines on cellular proliferation and differentiation is not unique to these compounds, having been observed for endogenous molecules such as leukotriene B440, BMPs41, the growth factors EGF/FGF242, and NGF/BDNF/bFGF43, and the transcription factors Lmx1a and Lmx1b44. However, the fact that the β-carbolines also stimulated migration highlights the versatility of these exogenous compounds, as they can promote the three processes involved in full adult neurogenesis.

A likely possible explanation for the observed effects of β-carbolines in neurogenesis is the increase in monoamine levels caused by MAO inhibition. With this said, we must acknowledge that the magnitude of the neurogenic effects was similar for the four compounds, despite harmol and THH being inhibitors that are between a hundred and a thousand times weaker than harmine or harmaline5. Moreover, the role of monoamines in neurogenesis is not fully understood. Knocking out the 5-HT 1A receptor in mice impaired neurogenesis after fluoxetine but not after imipramine, indicating that neurogenesis was independent from elevated serotonin levels45. In another study, the authors reported the unexpected finding that serotonin depletion actually promoted hippocampal neurogenesis instead of decreasing it46. In a recent paper, harmine, but not the MAO inhibitor pargyline, stimulated proliferation of human neural progenitor cells in vitro 33. Harmine effects were mediated through inhibition of the DYRK1A kinase rather than through MAO inhibition. This opens the possibility that the β-carbolines tested here regulated stem cell fate via DYRK1A or other alternative mechanisms. To our knowledge, the inhibitory effects of harmaline, tetrahydroharmine and harmol on DYRK1A has not been examined. Other potential molecular targets for the neurogenic effects of small molecules include the modulation of the GSK-3β/β-catenin pathway 47, upregulation of the brain-derived neurotrophic factor48, increased levels of vascular endothelial growth factor49; and glucocorticoid receptor activation50. Future studies should assess whether B. caapi β-carbolines interact with one or more of these pathways.

Our findings have relevant therapeutic implications. The association between neurogenesis and anti-depressant activity is well documented45, 51. Enhanced hippocampal neurogenesis reduces depression-like behaviors in animals51. Furthermore, clinically effective antidepressants stimulate neurogenesis, independent of their chemical structure and mechanism of action. To cite a few examples, chronic treatment with the serotonin reuptake inhibitor fluoxetine increases neurogenesis in rats52, 53, as does chronic treatment with the selective MAO-A inhibitor pirlindole53. The association between neurogenesis and antidepressant effect is not limited to rodents and pharmacological interventions. Electroconvulsive therapy in primates also stimulates proliferation of neural precursors in the hippocampus and their differentiation into neurons54. Hippocampal neurogenesis appears to be necessary for antidepressant action. Irradiation of the SGZ of the hippocampus in mice prevents the neurogenic and behavioral effects of fluoxetine and imipramine45.

In humans, two recent clinical studies have demonstrated rapid and long-lasting antidepressant effects after a single ayahuasca dose in patients who did not respond to conventional treatment11, 12. The therapeutic potential of ayahuasca is an area of increasing research interest beyond depression55. Alterations in adult neurogenic niches have been associated with a number of pathologies affecting the central nervous system56,57,58,59. Stimulation of these niches is currently being investigated as a novel therapeutic strategy for neuropsychiatric disorders60,61,62. Regular ayahuasca use has been associated decreases in problematic alcohol, cocaine and opiate consumption, indicating anti-addiction properties for B. caapi preparations63, 64. These potential anti-addictive properties are particularly relevant if we acknowledge the notorious difficulty of treating substance use disorders. Drug-dependent patients not only show functional deficits in reward processing and cognitive control, but also structural alterations in brain gray and white matter65.

Our study has a series of limitations that need to be acknowledged. Ayahuasca brews contain other active compounds that were not tested here. A popular version of ayahuasca in the USA and Europe contains DMT, a serotonergic psychedelic9. It is possible that DMT may have contributed to the antidepressant effects reported in clinical studies using ayahuasca11, 12. This contribution could be due to both brain plasticity mediated by 5-HT 2A receptor activation66 and to the profound psychological experiences induced by psychedelics67. While studying DMT in the neurogenesis model was not an objective of the present investigation, it could be assessed in a future study, comparing it with other 5-HT 2A agonists such as psilocybin or LSD. Although several animal studies have already shown that harmine improves behavioral measures of depression17, 18, future studies could ideally test the four compounds assessed here for both in vivo neurogenesis and behavioral improvement. Finally, future research could also use positive controls to compare the potency of the B. caapi β-carbolines with that of other antidepressants, such as SSRIs and MAO inhibitors.

In conclusion, here we showed that the β-carboline alkaloids present in B. caapi, the plant source of the ayahuasca tea, promote neurogenesis in vitro by stimulating neural progenitor pool expansion, and by inducing cellular migration and differentiation into a neuronal phenotype. The stimulation of neurogenic niches in the adult brain may substantially contribute to the antidepressant effects reported for ayahuasca in recent clinical studies. The versatility and full neurogenic capacity of the B. caapi β-carbolines warrant further investigation of these compounds. Their ability to modulate brain plasticity indicates their therapeutic potential for a broad range of psychiatric and neurologic disorders.