Author’s Accepted Manuscript
Inhibition of alpha oscillations through serotonin 2A
receptor activation underlies the visual effects of
ayahuasca in humans
Marta Valle, Ana Elda Maqueda, Mireia Rabella,
Aina Rodríguez-Pujadas, Rosa Maria Antonijoan,
Sergio Romero, Joan Francesc Alonso, Miquel
Àngel Mañanas, Steven Barker, Pablo Friedlander
Msc, Amanda Feilding, Jordi Riba
To appear in:
Received date: 2 August 2015
2 March 2016
Accepted date: 19 March 2016
Cite this article as: Marta Valle, Ana Elda Maqueda, Mireia Rabella, Aina
Rodríguez-Pujadas, Rosa Maria Antonijoan, Sergio Romero, Joan Francesc
Alonso, Miquel Àngel Mañanas, Steven Barker, Pablo Friedlander Msc, Amanda
Feilding and Jordi Riba, Inhibition of alpha oscillations through serotonin 2A
receptor activation underlies the visual effects of ayahuasca in humans,
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Inhibition of alpha oscillations through serotonin 2A receptor
activation underlies the visual effects of ayahuasca in humans
Ketanserin and ayahuasca
Marta Valle PhD
, Ana Elda Maqueda MSc
, Mireia Rabella MSc
, Rosa Maria Antonijoan PhD
, Sergio Romero PhD
Francesc Alonso PhD
, Miquel Àngel Mañanas PhD
, Steven Barker PhD
Pablo Friedlander Msc
, Amanda Feilding MSc
, Jordi Riba PhD
Pharmacokinetic and Pharmacodynamic Modelling and Simulation, IIB Sant Pau. Sant
Antoni María Claret, 167, 08025 Barcelona, Spain.
Centre d’Investigació de Medicaments, Servei de Farmacologia Clínica, Hospital de la
Santa Creu i Sant Pau. Sant Antoni María Claret, 167, 08025, Barcelona, Spain.
Department of Pharmacology and Therapeutics, Universitat Autònoma de Barcelona
(UAB), Barcelona, Spain.
Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain.
Human Neuropsychopharmacology Group. Sant Pau Institute of Biomedical Research
(IIB-Sant Pau). SantAntoni María Claret, 167. 08025, Barcelona, Spain.
Servei de Psiquiatria, Hospital de la Santa Creu i Sant Pau. SantAntoniMaría Claret,
167. 08025, Barcelona, Spain.
Biomedical Engineering Research Centre (CREB), Department of Automatic Control
(ESAII), Universitat Politècnica de Catalunya (UPC), Barcelona, Spain.
CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain.
Barcelona College of Industrial Engineering (EUETIB), UniversitatPolitècnica de
Catalunya (UPC), Barcelona08028, Spain.
Department of Comparative Biomedical Sciences, School of Veterinary Medicine,
Louisiana State University, Skip Bertman Drive at River Road, Baton Rouge, LA
The Beckley Foundation, Beckley Park, Oxford OX3 9SY, United Kingdom
Correspondence to: Jordi Riba. Human Neuropsychopharmacology Group, IIB-Sant
Pau. Sant Antoni María Claret, 167.08025, Barcelona, Spain. Phone: +34 93 556 5518.
Fax: +34 93 553 7855. Email: firstname.lastname@example.org
Ayahuasca is an Amazonian psychotropic plant tea typically obtained from two
plants, Banisteriopsis caapi and Psychotria viridis. It contains the psychedelic 5-HT
and sigma-1 agonist N,N-dimethyltryptamine (DMT) plus β-carboline alkaloids with
monoamine-oxidase (MAO)-inhibiting properties. Although the psychoactive effects of
ayahuasca have commonly been attributed solely to agonism at the 5-HT
molecular target of classical psychedelics, this has not been tested experimentally. Here
we wished to study the contribution of the 5-HT
receptor to the neurophysiological
and psychological effects of ayahuasca in humans. We measured drug-induced changes
in spontaneous brain oscillations and subjective effects in a double-blind randomized
placebo-controlled study involving the oral administration of ayahuasca (0.75 mg
DMT/kg body weight) and the 5-HT
antagonist ketanserin (40 mg). Twelve healthy,
experienced psychedelic users (5 females) participated in four experimental sessions in
placebo+ayahuasca, ketanserin+placebo and ketanserin+ayahuasca. Ayahuasca induced
EEG power decreases in the delta, theta and alpha frequency bands. Current density in
alpha-band oscillations in parietal and occipital cortex was inversely correlated with the
intensity of visual imagery induced by ayahuasca. Pretreatment with ketanserin
inhibited neurophysiological modifications, reduced the correlation between alpha and
visual effects, and attenuated the intensity of the subjective experience. These findings
suggest that despite the chemical complexity of ayahuasca, 5-HT
activation plays a
key role in the neurophysiological and visual effects of ayahuasca in humans.
Key words: Ayahuasca, serotonin-
receptor, ketanserin, subjective effects,
neurophysiological effects, human
Ayahuasca is a psychoactive plant tea used traditionally by the indigenous
peoples of the Upper Amazon (Schultes, 1980) and in more recent times by healers and
members of religious syncretic groups (Tupper, 2008). This tea is receiving increased
attention from the general public and biomedical researchers (Frood, 2015). It has been
used to help treat addiction (Fernández et al., 2014), and recent open-label studies have
shown preliminary evidence of rapid and lasting antidepressant effects after a single
dose (Osório et al., 2015; Sanches et al., 2015).
Although there are many variations in the preparation of the tea, the common
ingredient is the malpighiaceous vine Banisteriopsis caapi. This plant is rich in β-
carboline alkaloids, mainly harmine, harmaline and tetrahydroharmine (THH) (Riba,
2003). These alkaloids show monoamine-oxidase inhibiting properties (N S Buckholtz
and Boggan, 1977), while THH is also a serotonin reuptake inhibitor (N. S. Buckholtz
and Boggan, 1977). In addition to B. caapi, other admixture plants are frequently used
in the preparation of ayahuasca. One of the most common in the context of modern use
is Psychotria viridis. The leaves of this plant are rich in the psychedelic indole N,N-
dimethyltryptamine or DMT (Riba, 2003).
DMT is structurally related to the neurotransmitter serotonin (5-
hydroxytryptamine; 5-HT) and shows agonist activity at the 5-HT
receptors. DMT also acts as an agonist at the trace amine associated receptor (TAAR)
(Bunzow et al., 2001) and it is a substrate of the serotonin and the vesicle monoamine
transporters (Cozzi et al., 2009). It has been suggested that using these uptake
mechanisms, intracellular concentrations could reach higher values than in plasma and
interact with the intracellular sigma-1 receptor (Fontanilla et al., 2009). This receptor
modulates the activity of many other proteins, conferring stability against cellular stress,
and promoting brain plasticity (Chu and Ruoho, 2016; Tsai et al., 2009).
When administered to humans parenterally, DMT induces intense modifications
of the ordinary state of awareness with intense visual effects, but it is devoid of
psychoactivity when taken orally (Riba et al., 2015) due to degradation by MAO
(Suzuki et al., 1981), and cytochrome-dependent mechanisms (Riba et al., 2015). The
presence of the MAO-inhibiting β-carbolines in ayahuasca prevents is enzymatic
degradation and allows its oral bioavailability (Riba et al., 2003a).
In previous studies by our group, we found ayahuasca to induce a pattern of
psychedelic effects with a slower onset and longer duration than those induced by DMT
(Dos Santos et al., 2011; Riba et al., 2003a, 2001b). Neurophysiologically, ayahuasca
induces broad-band power decreases in spontaneous electrical brain oscillations (Riba et
al., 2002a) and associated reductions in intracerebral current source density (CSD) in
certain brain areas (Riba et al., 2004). These reductions are particularly strong for
oscillations in the alpha band of the EEG, with CSD reductions over the posterior visual
cortex, an effect thought to reflect increased cortical excitability (Romei et al., 2008b).
Analogous findings in the range of the alpha band have also been observed using
magnetoencephalography and the psychedelic and serotonin-
psilocybin (Muthukumaraswamy et al., 2013).
The aim of this study was to assess the contribution of serotonin-
the neurophysiological and psychological effects of ayahuasca. We postulated that
despite the combination of various pharmacological mechanisms in ayahuasca, the
general psychedelic effects and decreases in current density depend on activation of the
receptor. To test this hypothesis, we studied the interaction of a medium dose of
ayahuasca (Riba et al., 2001b) and ketanserin, a 5-HT
receptor antagonist, in a group
of experienced psychedelic users in a laboratory setting.
For ethical reasons, we only recruited individuals with prior experience with
psychedelics. We wanted to avoid introducing drug-naive individuals to psychedelics,
and to make sure that volunteers would be familiar with the modified state of
consciousness induced by these drugs. We therefore contacted psychedelic drug users
and informed them about the goals of the study, the nature of ayahuasca, its
psychological effects, and the potential adverse effects described in the literature for
psychedelics. We recruited a group of 12 healthy volunteers (5 females, 7 males) with
previous experience with psychedelic drugs (10 times or more). Despite their experience
with psychoactive substances, no participant had a current or previous DSM/ICD-10
diagnosis of drug dependence.
The volunteers had a mean age of 35 years (26-43). Their past experience with
psychedelic drugs mainly involved LSD (11/12), Psilocybe mushrooms (11/12) and
ayahuasca (8/12). Nine of the participants also had experience with ketamine, six had
used 2C-B, five had smoked Salvia divinorum, four had taken mescaline-containing
cacti such as peyote or San Pedro, and two had smoked dimethyltryptamine. At the time
of the study, eight were using cannabis sporadically (1-2 cigarettes per week). Only four
were currently tobacco smokers and eleven consumed alcohol in moderate amounts,
from one or two beers or glasses of wine per day to one per month.
Prior to participation, all volunteers underwent a complete medical examination
that included medical history, physical examination, ECG, and standard laboratory tests,
to confirm good health. Exclusion criteria included a current or past history of
psychiatric disorders, alcohol or other substance use disorders, evidence of significant
illness, and pregnancy. The study was conducted in accordance with the Declaration of
Helsinki and subsequent amendments concerning research in humans and was approved
by the Sant Pau Hospital Ethics Committee and the Spanish Ministry of Health. All
volunteers gave their written informed consent to participate.
Ayahuasca was administered in freeze-dried encapsulated form. The ayahuasca
batch used in the study was analyzed using a previously described method using liquid
cromatography-electrospray ionization-tandem mass spectrometry (McIlhenny et al.,
2009). The analysis showed that ayahuasca contained the following alkaloid
concentrations in mg per gram of freeze-dried material: 6.51 DMT, 13.14 harmine, 1.35
harmaline and 11.55 THH. The final dose was calculated individually for each
participant, so that they received the equivalent of 0.75 mg DMT/kg body weight. The
dose chosen is of medium intensity and was selected based on data from previous
studies where it showed robust psychological and physiological effects (Dos Santos et
al., 2012; Riba et al., 2001b). Given the alkaloid proportions present in the freeze-dried
material, at the 0.75 mg/kg DMT dose, participants also ingested 1.51 mg/kg of
harmine, 0.16 mg/kg of harmaline and 1.33 mg/kg of THH.
Ketanserin was administered as the trademark drug Ketensin (ketanserin
tartrate), at the dose of 40 mg, and placebo capsules contained lactose.
Study design and drug administration
The study was conducted according to a double-blind, randomized, balanced,
crossover design. It involved four experimental sessions one week apart each. Two
weeks prior to the first experimental session and throughout the study, participants
abstained from any psychoactive drugs and medications. Urine was collected for drug
analysis on each experimental day. Participants tested negative for alcohol, cannabis,
amphetamines, benzodiazepines, opiates and cocaine. In each session, participants
received an initial treatment that could be placebo (lactose capsule) or 40 mg ketanserin.
One hour later, they were administered a second placebo or encapsulated freeze-dried
ayahuasca. Thus, on each experimental day, participants received one of four different
treatment combinations: placebo+placebo, placebo+ayahuasca, ketanserin+placebo and
Participants remained in the laboratory for 8 hours after which they were
discharged home. During the first four hours they remained seated in a reclining chair in
a sound-attenuated and dimly lit room. EEG recordings were conducted before the
administration of the first treatment (placebo or ketanserin). Ninety minutes after
administration of the second treatment (ayahuasca or placebo), when the peak
ayahuasca effects were expected, a second EEG recording was obtained. Four hours
after administration of the second treatment, when most of the subjective effects of
ayahuasca had disappeared, the volunteers were allowed to leave the room and were
asked to answer the subjective effects questionnaires.
EEG recording and processing
Three-minute EEG recordings with eyes closed were obtained from 19 standard
scalp locations (Fp1/2, F3/4, Fz, F7/8, C3/4, Cz, T3/4, T5/6, P3/4, Pz and O1/2).
Recordings were obtained using a BrainAmp amplifier (Brain Products GmbH,
Gilching, Germany) before the first treatment (baseline) and 90 minutes after
administration of the second treatment. Signals were referenced to the averaged mastoid
electrodes, and vertical and horizontal electrooculograms (EOG) were also obtained for
artifact minimization and removal. Signals were analogically band-pass filtered between
0.1 and 45 Hz, digitized with a frequency of 250 Hz.
EEG artifact minimization and removal was performed according to a two-step
procedure before calculating the parameters. First, an ocular artifact minimization step
was implemented using a previously described method based on blind source separation
or BSS (Alonso et al., 2010; Romero et al., 2008). The continuous EEG recording was
then segmented into 5 second epochs. These segments were automatically analyzed for
saturation, muscular and movement artifacts using the procedure described by Anderer
and colleagues (Anderer et al., 1992).
After computing the two-step artifact preprocessing procedure, spectral analysis
was performed for all EEG channels. Power spectral density (PSD) functions were
calculated from artifact-free 5 second epochs by means of a periodogram using a
Hanning window, and averaged. Averaged PSD functions for each experimental
situation were quantified into absolute powers in the following frequency bands: delta
(0.5–3.5 Hz), theta (3.5–7.5 Hz), alpha (7.5-13 Hz), and beta (13-35 Hz). Additionally,
frequency variability was measured calculating the deviation of the center-of-gravity
frequency or centroid of the total activity (0.5-35 Hz).
Intracerebral current density calculation
The Standardized LORETA (sLORETA) software (Pascual-Marqui et al., 1994)
was used to estimate the three-dimensional intracerebral current density distribution
from the voltage values recorded at the scalp. sLORETA estimates a particular solution
of the non-unique EEG inverse solution restricted to 6239 cortical grey matter voxels
with a spatial resolution of 0.125 cm
according to a digitized head model from
Montreal Neurological Institute (Pasqual-Marqui, 2002). The current density values
were estimated based on the EEG cross-spectral matrix and then squared for each voxel
in the classical frequency bands.
Subjective effect measures
The psychological effects elicited by the administered treatments were measured
using a battery of questionnaires on subjective effects: the Hallucinogen Rating Scale
(HRS), the Addiction Research Center Inventory (ARCI), the Altered States of
Consciousness Questionnaire (APZ), and a battery of self-administered visual analogue
The Hallucinogen Rating Scale (HRS), developed by Strassman and colleagues
(Strassman et al., 1994), includes 71 items grouped in six subscales: Somaesthesia,
reflecting somatic effects; Affect, measuring emotional and affective responses;
Perception, measuring visual, auditory, gustatory, and olfactory experiences; Cognition,
describing modifications in thought processes or content; Volition, indicating the
volunteer's capacity to willfully interact with his/her “self” and/or the environment; and
Intensity, which reflects the strength of the overall experience. The range of scores for
all scales is 0-4. A validated Spanish version was administered (Riba et al., 2001a).
The Addiction Research Center Inventory (ARCI) (Martin et al., 1971) includes
49 items distributed in five scales or groups: the morphine-benzedrine group (MBG)
that measures euphoria; the pentobarbital-chlorpromazine-alcohol group (PCAG), that
measures sedation; the lysergic acid diethylamide scale (LSD), that measures somatic-
dysphoric effects; the benzedrine group (BG) that measures subjectively experienced
intellectual efficiency; and the amphetamine scale (A), which is sensitive to stimulants.
The range of scores is 0-16 for MBG, -4 to 11 for PCAG, -4 to 10 for LSD, -4 to 9 for
BG, and 0-11 for A. A validated Spanish version was administered (Lamas et al., 1994).
The Altered States of Consciousness questionnaire (“Aussergewöhnliche
Psychische Zustände”, APZ) (Dittrich, 1998) is composed of 72 items distributed in
three subscales: Oceanic Boundlessness (“Ozeanische Selbst-entgrenzung”, OSE), to
measure changes in the sense of time, derealization and depersonalization; Dread of