Abstract
N,N-dimethyltryptamine (DMT), a psychedelic compound identified endogenously in mammals, is biosynthesized by aromatic-L-amino acid decarboxylase (AADC) and indolethylamine-N-methyltransferase (INMT). Whether DMT is biosynthesized in the mammalian brain is unknown. We investigated brain expression of INMT transcript in rats and humans, co-expression of INMT and AADC mRNA in rat brain and periphery, and brain concentrations of DMT in rats. INMT transcripts were identified in the cerebral cortex, pineal gland, and choroid plexus of both rats and humans via in situ hybridization. Notably, INMT mRNA was colocalized with AADC transcript in rat brain tissues, in contrast to rat peripheral tissues where there existed little overlapping expression of INMT with AADC transcripts. Additionally, extracellular concentrations of DMT in the cerebral cortex of normal behaving rats, with or without the pineal gland, were similar to those of canonical monoamine neurotransmitters including serotonin. A significant increase of DMT levels in the rat visual cortex was observed following induction of experimental cardiac arrest, a finding independent of an intact pineal gland. These results show for the first time that the rat brain is capable of synthesizing and releasing DMT at concentrations comparable to known monoamine neurotransmitters and raise the possibility that this phenomenon may occur similarly in human brains.
Introduction
N,N-dimethyltryptamine (DMT) belongs to a class of serotonergic psychedelics that includes lysergic acid diethylamide (LSD) and psilocybin1. DMT, like all serotonergic psychedelics, reliably elicits a wide spectrum of subjective effects on brain functions including perception, affect, and cognition2. These compounds share structural and functional similarities with serotonin (5-hydroxytryptamine, 5-HT), and interact with 5-HT and other receptors to produce their effects3,4,5. Unlike other psychedelics, however, DMT is endogenously produced in animals6,7,8, including humans9,10,11. In addition to the subjective psychedelic effects exogenous administration of DMT has on conscious experience, it has other well-documented anti-hypoxic12, antidepressant13, and plasticity-promoting actions14. Taking these facts together, a further understanding of why DMT is present in mammals is of interest.
Biosynthesis of DMT from tryptamine requires double methylation reactions catalyzed by indolethylamine-N-methyltransferase (INMT)15,16. INMT mRNA was identified at high levels in peripheral tissues in rabbits17 and in humans18. However, this peripheral INMT also methylates other ligands such as histamine17,19. In the brain, INMT mRNA was found at very low levels in rabbits17 and was undetectable in humans18. No study to date has yet identified INMT in the cerebral cortex in any species. In addition to INMT, production of DMT requires aromatic-L-amino acid decarboxylase (AADC), which removes the carboxyl group from dietary tryptophan to form tryptamine, the essential DMT precursor that can be rapidly metabolized by monoamine oxidase20. While high levels of INMT mRNA expression in the periphery17,18 have been assumed to indicate the potential for correspondingly high levels of DMT, cellular colocalization of INMT and AADC transcripts has not yet been reported in any tissue. Moreover, studies to date assessing levels of DMT in human peripheral bodily fluids have only reported it in trace amounts11, calling into question any physiological role.
In order to address some of these issues, this study examined expression of INMT mRNA in rat and human brain tissues using in situ hybridization. In addition, we conducted double in situ hybridization studies to probe the co-expression of INMT and AADC mRNA in rat brain and periphery. To investigate the presence of DMT in rat brain, we analyzed DMT content in microdialysis samples collected directly from the visual cortices of living pineal-intact and pinealectomized rats. Pinealectomized animals were studied because previous data established the presence of DMT in living rat pineal dialysate8. To investigate whether DMT brain levels are inducible by physiological alterations, we assessed its levels in rat brain following cardiac arrest with or without the pineal gland, as a prior study from our lab demonstrated a surge in the levels of select neurotransmitters in rat visual cortex following cardiac arrest using this technique21.
...
Discussion
We report [1] cortical expression of INMT mRNA in rat and human brain, [2] colocalization of INMT and AADC mRNA in the same cells in rat brain, and [3] predominately non-overlapping expression of INMT with AADC mRNA in rat peripheral organs including the adrenal, kidney, lung, and heart. We further show that DMT is present in rat visual cortex in pineal-intact and pinealectomized animals. Moreover, we show DMT levels are significantly elevated by experimentally-induced cardiac arrest. Collectively, these data support the notion that DMT is synthesized in rat brain and at concentrations consistent with that of other known monoamine neurotransmitters. Our demonstration of INMT mRNA expression in human cerebral cortex, choroid plexus, and pineal gland also suggest that DMT biosynthesis may similarly occur in the human brain.
This study demonstrates for the first time that INMT mRNA, the transcript for a key DMT synthetic enzyme, is widely expressed in the cerebral cortex in rats. Importantly, INMT and AADC transcripts are co-expressed in the same brain cells, providing a plausible mechanism for cellular synthesis of DMT in the mammalian neocortex. DMT is a monoamine produced from tryptophan. If endogenous DMT functions as a non-classical monoamine neurotransmitter in the brain, DMT would be the only monoamine whose biosynthesis takes place within the cerebral cortex where it may directly influence cognitive functions of the brain.
While AADC expression in the brain is well known26, INMT expression in the brain was unclear prior to our study. Earlier studies showed that INMT mRNA was only weakly present in rabbit brain tissues17, and undetectable in human brain tissues18. These prior reports are all based on Northern blot analysis, which provides no information on cellular distribution of INMT mRNA and is less sensitive compared to quantitative polymerase chain reaction or the in situ hybridization method used in our study. We were able to identify INMT mRNA in both rat and human brain tissues using the RNAscope in situ assay system, in part because of the high sensitivity afforded by this technique27, which allowed robust and unambiguous identification of INMT mRNA in both rat and human brain FFPE tissues for the first time (see Fig. 1).
AADC is best characterized as one of the enzymes necessary for the synthesis of all canonical monoamine neurotransmitters, including 5-HT [together with tryptophan hydroxylase (TPH)] and dopamine/norepinephrine [together with tyrosine hydroxylase (TH)]. In the pineal gland, AADC (together with TPH) is well known to mediate the synthesis of 5-HT, the precursor for melatonin production28. In the brain, AADC mRNA is present abundantly in the monoamine neurons in the brainstem26. However, AADC mRNA and protein have also been reported in neurons of the cerebral cortex and hippocampus26 that do not contain TH/TPH in mice, rats and humans29,30,31,32,33. To date, the function of these AADC-positive and TH/TPH-negative neurons is unknown.
Using the RNAscope in situ hybridization technique, we identified AADC mRNA in several regions of the rat brain (Fig. 2), including the cerebral cortex and hippocampus (Fig. 2), consistent with what has been reported in mouse brain26. Critically, we found in each of these rat structures that AADC mRNA is colocalized with an INMT transcript (Fig. 2), suggesting that these INMT-positive and AADC-positive neurons may function as DMT-producing neurons (or D-neurons) and that DMT, a non-canonical monoamine, may possibly be the principle neurotransmitter in these widely distributed brain D-neurons. A functional role of the D-neurons will need to be explored in future studies. For example, INMT-deficient animals are necessary to demonstrate unequivocally that INMT is responsible for the endogenous production of DMT.
INMT protein was also found in monkey pineal gland in a prior study34. Our study expanded this finding and demonstrated an abundant expression of INMT mRNA in the pineal gland in both rats and humans (Fig. 1). Co-expression of INMT and AADC mRNA in rat pinealocytes was confirmed by double in situ hybridization analysis (Fig. 2), suggesting that a potential mechanism for DMT biosynthesis, as in the rat visual cortex, exists in the pineal gland. Transcripts for DMT synthetic enzymes were detected additionally in the choroid plexus, a venous network lining the ventricles of the brain that produce CSF, a fluid in which DMT has been detected11. The choroid plexus contains a high density of 5-HT2C receptors whose stimulation by agonists leads to marked reduction of CSF production35. As DMT is an agonist at the 5-HT2C receptor4,5,36 and LSD binds to the 5-HT2C receptor in cultured choroid plexus epithelial cells to stimulate their activity37, the effect of endogenous DMT on CSF regulation deserves additional investigation.
The high levels of INMT mRNA previously found in peripheral tissues18 have led to speculation that DMT is released from peripheral stores and subsequently transported to the brain. Consistent with earlier Northern blot analysis of human tissues18, we found high levels of INMT mRNA expression in rat kidney, lung, heart, and adrenal (Fig. 3). However, INMT mRNA expression in rat peripheral tissues showed very limited overlap with AADC mRNA at the cellular level, suggesting that INMT may have functions independent of DMT synthesis in the periphery. Although our present data suggest that DMT in the brain does not originate from peripheral sources but, rather, is produced locally in specific brain tissues, future studies (using brain-specific INMT knockout animals, for example) could be conducted to conclusively demonstrate that DMT found in brain dialysates originates from the brain. Peripheral INMT may be responsible for established DMT-independent functions such as methylation of sulfur-containing compounds38, histamine17,19, and selenium metabolism39.
DMT is an endogenous monoamine whose physiological functions remain unknown. Furthermore, since exogenous DMT can bind with nanomolar affinities to various receptors including 5-HT receptors40,41,42,43 and trace-amine associated receptors (reviewed in ref.44), endogenous DMT may influence brain functions via the same receptors. If DMT were indeed to function as a non-canonical monoamine neurotransmitter, however, it must be present in the brain at a physiologically relevant concentration. Presence of DMT has been reported in various species including rats6,7,8,9,10,11, yet its in vivo concentration in living brain has not been reported. This study represents the first quantification of DMT in the extracellular fluid of the brain in freely moving and normal behaving animals.
The baseline concentration of DMT in cortical microdialysates ranged between 0.05 to 1.8 nM (blue dots in Fig. 4Ab) in pineal-intact rats, and between 0.25 to 2.2 nM (blue dots in Fig. 4Bb) in pinealectomized rats. DMT levels showed no significant difference between rats with the pineal and without the pineal gland (Fig. 4Ca). Importantly, this cortical concentration of DMT (average 1.02 nM) is only slightly lower than that of 5-HT detected in the same dialysates, which averaged around 2 nM in rats without the pineal gland (Fig. 4Cc). This value of 5-HT in brain dialysates is consistent with what others have found in rats (average 0.87 nM; 0.12–3.4 nM range) [reviewed in ref.25]. In fact, the basal DMT concentrations (Fig. 4Ab; average 1.02 nM; 0.25–2.2 nM range) in the brain microdialysates are well within the known range of all three canonical monoamine neurotransmitters25: 5-HT (average 0.87 nM; 0.12–3.4 nM range), norepinephrine (average 1.77 nM; 0.19–4.4 nM range), and dopamine (average 1.5 nM; 0.07–4.9 nM range). We wish to emphasize that our microdialysates were collected and analyzed online in real time from rats without any pretreatment to block the activity of monoamine oxidase, an enzyme that rapidly degrades DMT in vivo15,45. The mechanism and function of cortical DMT production remain to be fully investigated.
DMT was previously detected in freely moving rats8. However, DMT was not quantified in that study. Furthermore, the microdialysates used for DMT analysis8 were collected from both the pineal gland and the surrounding visual cortex of rats and it was unclear whether the detected DMT was from the pineal alone, the cortex alone, or both. To address this latter issue, we surgically removed the pineal gland and monitored DMT levels in rats both under baseline and following cardiac arrest (Fig. 4B). We found that DMT concentrations in the brain are independent of the pineal gland, as cortical DMT levels show no significant difference with or without the pineal gland (Fig. 4Ca). These data suggest that the cortex may be one of the major sources of released DMT from the brain. At this point, it is uncertain to what extent, if any, the pineal contributes to DMT production8. It is also unclear whether DMT detected in the brain extracellular fluid is secreted from neurons and/or glial cells. Colocalization studies using both an INMT probe and cell-type specific markers should be used in future studies to clarify this.
The extensive colocalization of INMT and AADC transcripts in pinealocytes (Fig. 2C), and the apparent lack of the pineal’s contribution to the brain production of DMT (Fig. 4Ca) present a paradox. We reasoned that there may be two competing pathways for tryptophan in the pineal: (1) conversion of tryptophan to 5-HT via sequential actions of TPH and AADC, and (2) the potential conversion of tryptophan to DMT via sequential actions of AADC and INMT. It is plausible that in the pineal, the first pathway is overwhelmingly dominant, resulting in high production of 5-HT at the expense of DMT. The established properties of the enzymes support this: the affinity of tryptophan for TPH (Km = 41.3 uM)46 in the 5-HT/melatonin synthesis pathway is substantially higher compared to the reported affinity of tryptophan for AADC (Km = 3000 uM)47 in the DMT biosynthetic pathway. Thus, if the same cell expresses both TPH and AADC, tryptophan will be more likely to be converted to 5-hydroxytryptophyan (destined to 5-HT) than to tryptamine (destined to DMT). Additionally, 5-hydroxytryptophan binds to AADC with substantially higher affinity than tryptophan (Km = 160 uM48 versus Km = 3000 uM)47. As a result, high levels of 5-hydroxytryptophan found in the pineal are likely to competitively inhibit AADC conversion of tryptophan to tryptamine, further reducing this intermediate in DMT production. The relatively low affinity of AADC for tryptophan thus reduces the production of DMT in pineal cells that contain both TPH and AADC. These data may explain the possibility that the pineal gland contributes little to DMT production, despite the abundant co-expression of AADC and INMT in the pinealocytes. We could not, however, rule out the possibility that there may be compensatory increases in DMT’s biosynthesis in other brain areas following the loss of the pineal gland.
In our previous studies, we have observed a marked elevation of some, but not all, critical neurotransmitters in rat brain during asphyxic cardiac arrest21, which we posit may contribute to the elevated conscious information processing observed in dying rats21,49. These data also suggest that global ischemia (by cardiac arrest, as in the current study), similar to global hypoxia (by asphyxia, as in21), leads to a tightly regulated release of a select set of neurotransmitters21. To test whether DMT concentrations are regulated by physiological alterations, we monitored DMT levels in rat brain dialysates following experimentally-induced cardiac arrest, and identified a significant rise in DMT levels in animals with (Fig. 4A) and without the pineal (Fig. 4B).
The cardiac arrest-induced increase of endogenous DMT release may be related to near-death experiences (NDEs), as a recent study reports NDE-like mental states in human subjects given exogenous DMT50. Not all rats in our current study exhibited a surge of DMT following cardiac arrest (Fig. 4), an interesting observation in light of the fact that NDEs are reported by less than 20% of patients who survive cardiac arrests51. It is unknown whether the concentrations of DMT reported in our study at cardiac arrest can elicit the effects of an exogenous psychedelic dose of DMT, or whether this surge of endogenous DMT similarly occurs in humans. Moreover, the conscious states reported by NDE survivors may involve contributions from several of the other neurotransmitters found to surge at cardiac arrest in our prior rodent study21. Further investigation is clearly warranted to investigate whether DMT plays a role in generating neural correlates of near-death consciousness.
In conclusion, we present evidence for the brain expression of mRNA for INMT, the key DMT synthetic enzyme previously thought to exist only in the periphery, and demonstrate that, unlike in the periphery, transcripts for the DMT synthetic enzymes AADC and INMT are co-expressed in the same cells within the rat brain. The wide co-expression of transcripts for the DMT synthetic enzymes in the rat brain and basal concentrations of DMT comparable to that of other monoamine neurotransmitters indicate that endogenous DMT may influence brain function.
...
www.nature.com
