REVIEW ARTICLE
Fungal Hallucinogens Psilocin, Ibotenic Acid, and
Muscimol: Analytical Methods and Biologic Activities
Katarzyna Stebelska, PhD
Abstract: Psychoactive drugs of fungal origin, psilocin, ibotenic
acid, and muscimol among them have been proposed for recreational
use and popularized since the 1960s, XX century. Despite their well-
documented neurotoxicity, they reached reputation of being safe and
nonaddictive. Scientific efforts to find any medical application for
these hallucinogens in psychiatry, psychotherapy, and even for
religious rituals support are highly controversial. Even if they show
any healing potential, their usage in psychotherapy is in some cases
inadequate and may additionally harm seriously suffering patients.
Hallucinogens are thought to reduce cognitive functions. However,
in case of indolealkylamines, such as psilocin, some recent findings
suggest their ability to improve perception and mental skills, what
would motivate the consumption of “magic mushrooms.” The pres-
ent article offers an opportunity to find out what are the main symp-
toms of intoxication with mushrooms containing psilocybin/psilocin,
muscimol, and ibotenic acid. The progress in analytical methods
for detection of them in fungal material, food, and body fluids is
reviewed. Findings on the mechanisms of their biologic activity are
summarized. Additionally, therapeutic potential of these fungal psy-
choactive compounds and health risk associated with their abuse are
discussed.
Key Words: hallucinogens, psilocin, ibotenic acid, muscimol
(Ther Drug Monit 2013;35:420–442)
INTRODUCTION
Psychoactive plants or fungi accompanied religious rituals
in many ancient civilizations worldwide.1–4 Hallucinogenic com-
pounds contained in fungi of Psilocybe and Amanita genera
have been used as recreational drugs of abuse since the begin-
ning of the 1960s, XX century.5,6 Although hallucinogens are
thought to be safe because of relatively low physiologic tox-
icity and dependence potential7,8 nowadays the context of
hallucinogenic compounds consumption, mainly by young
people seeking for unusual experiences, is usually nega-
tive5,6,9 but not exclusively. One exception seems to be the
Received for publication June 19, 2012; accepted January 9, 2013.
From the Department of Analytical and Ecological Chemistry, Opole University,
Opole, Poland.
Supported by Wroclaw Research Centre EIT+ under the Project “Biotechnologies
and advanced medical Technologies”—BioMed (POIG.01.01.02-02-003/08)
financed from the European Regional Development Fund (Operational Pro-
gramme Innovative Economy, 1.1.2).
The author declares no conflicts of interest.
Correspondence: Katarzyna Stebelska, PhD, Department of Analytical and
Ecological Chemistry, Opole University, Pl Kopernika 11, PL-45-040
Opole, Poland (e-mail: kasia.stebelska@op.pl).
Copyright © 2013 by Lippincott Williams & Wilkins
420
idea to use them for religious ceremonies or psychotherapy
support. As an example, it was experimentally established
that psilocybin, psilocin prodrug of fungal origin, is able to
produce mystical states under favorable circumstances
(involving acquired cultural frame of references).10–16 Some
details of the study design, particularly the usage of niacin as
an active placebo, may suggest that the authors of the first
of these experiments known as Good Friday Experiment
were aware that religious rituals alone are not neutral for
mental health.10–28 The states of altered consciousness induced
by religious settings and facilitated by psychedelic drugs
increase susceptibility to suggested messages and are not free
from negative emotions such as fear, terror, anxiety, shame,
and sense of guilt. Therefore, any religious cult, particularly
if combined with psychoactive compounds intake, should
be considered as being able to alter psychosocial identity.
Psychotic syndromes evoked in religious purposes are not
fully safe and may induce development of really serious psy-
chopathologies including confusional syndrome and schizo-
phrenia, a disease of at least partly psychosocial etiology.29–35
It should be pointed out that psilocybin/psilocin, although still
scientifically explored as potentially helpful and safe psycho-
therapy adjunctive,4,10–16,36–40 is presently used to model
schizophrenia and to investigate psychosis similar to positive
symptoms of the disease.41–50
The psychoactive principles of Psilocybe mushrooms,
namely psilocybin and psilocin, were isolated and identified
by Hofmann in 1958.51 Both these substances, similarly as
LSD, were originally proposed as incapacitating agents and
used for intelligence purposes.7 Ibotenic acid and muscimol
seemed to be the most active substances of fly agaric respon-
sible for its hallucinogenic properties.52–54 Exposure to these
substances, especially chronic, not only may constitute a sig-
nificant risk to human mental health but may also induce
other serious disorders including cardiovascular complica-
tions and neurodegenerative diseases. Although chemically
unstable, ibotenic acid should be considered as highly toxic
agent if one is aware of convulsant activity and excitotoxicity
of its endogenous analogue glutamate. Simultaneously, the
fungal psychedelics of high biologic activity display signifi-
cant therapeutic potential.
Hallucinogenic fungi and psychedelics isolated from
them are illegal in many countries or their processing and
distribution stay under strict regulation.12 However, because
they are easily available from natural habitats, they have
become quite popular among growing population of drug
users and are frequent cause of serious poisoning or even
accidental deaths resulted from their biologic activity.5–9,55–59
Ther Drug Monit  Volume 35, Number 4, August 2013
Ther Drug Monit  Volume 35, Number 4, August 2013
Therefore, analytical methods elaborated for detection of these
substances in fungal material and body fluids are also reviewed
in this article.
Psilocybin/Psilocin as a Secondary Metabolite
Psilocybin is a secondary metabolite of some species of
Agrocybe, Conocybe, Copelandia, Galerina, Gymnopilus,
Hypholoma, Inocybe, Panaeolina, Panaeolus, Pholiotina,
Pluteus, and Psilocybe genera.60–63 The major part of the
known psychoactive species belongs to the genus Psilocybe.
They are saprotrophic on wood, dung, moss, soil, and litter.
Psilocybe cyanescens, P. serbica var. bohemica, P. serbica
var. arcana, and P. semilanceata are probably the only spe-
cies containing sufficient for intoxication amounts of psilocin/
psilocybin that can grow in natural or near natural habitats in
the Middle and North Europe.60,64–66 Psilocybe cubensis is the
most popular natural source of psilocybin in the warmer
regions of the Americas. In the United States, it can be found
in the wild throughout the southeast to Texas and the Pacific
Northwest. The fungus is common in Mexico and other Cen-
tral American countries, and it is also known from South
American, Asian, Australian and Oceanian localities.62,67,68
Psilocybe subaeruginosa occurs commonly in Australia and
is widespread in New Zealand and Tasmania.68,69
Season-dependent availability of psilocybin-containing
mushrooms has stimulated the search for methods to cultivate
these fungi. Culturing of the mushrooms became common
after reports on the methodology seemed in both scientific
literature and specialist books. It seems that most recreation-
ally used mushrooms are cultivated rather than picked wild at
present. For illicit home growers, P. cubensis and its varieties
are generally the mushrooms of choice because they are easy
to culture and fruit readily in vitro, producing a significant
amount of biomass with each flush.60,67
Alkaloids isolated from hallucinogenic mushrooms
within Psilocybe genus such as psilocybin, baeocystin, nor-
baeocystin are tryptophan derivatives formed through decar-
boxylation, indole ring hydroxylation at position 4, and
N-methylation through the activity of S-adenosylmethionine
and O-phosphorylation. Biosynthetic pathway of psilocybin
in P. cubensis involves conversion of tryptophan to trypt-
amine, N-methyltryptamine, N,N-dimethyltryptamine, and
psilocin or alternatively conversion of 4-hydroxytryptamine
to psilocybin.70 Psilocybe cubensis cultures have the ability
to hydroxylate tryptamine derivatives at position 4.71
Psilocybe spores and monocaryotic mycelium do not
contain psychoactive compounds. Mycelium starts to produce
psilocybin/psilocin at further stages of mushroom develop-
ment.72 Quantitative analysis of P. cubensis fruit bodies col-
lected from natural sources or from artificial cultures grown
under strictly controlled conditions reveals that the content of
psilocin varies greatly (eg, from a 3.2–13.3 mg/g for artificial
culture).73 The first flush collected from culture cultivated on
rice grain generally does not contain psilocin.73
The amount of psilocybin depends on composition of
nutrient medium. Media depleted in source of carbon are less
effective in the alkaloid production.74,75 Tryptophan supple-
mentation stimulates psilocybin biosynthesis. This happens

2013 Lippincott Williams & Wilkins
Psilocin, Ibotenic Acid, and Muscimol
when tryptophan is present in the medium from the stage of
culture inoculation.75 Replacement of nutrient medium does
not affect psilocybin content.74 Conditions of potassium
depletion do not influence considerably mycelium growth
but lower psilocybin production.75 Lack of succinate in the
medium decreases biosynthesis of psilocybin.74
Psilocybin seems to be present at higher amounts in
mushroom caps than in the stems.76,77 Psilocybe semilanceata
and P. serbica var. bohemica (synonymous with P. bohemica)
were found to contain high amounts of psilocybin up to 1% of
dry weight. Psilocybe cubensis seemed to contain minor
amounts of psilocybin and low amounts of psilocin (0.63%
and 0.11%, respectively). The contents of psilocybin and psi-
locin in the sample of P. cyanescens were 0.32% and 0.51%,
respectively.78 Psilocybe serbica var. bohemica collected in
Europe were found to contain minor amounts of psilocybin
(content between 0.33% and 0.62%) and almost no psilocin.
Psilocybe cyanescens from the Pacific Northwest, United
States, contained both psilocybin (0.41%–0.98%) and psilocin
(0.23%–0.93%).79
During mycelium development, some processes are
controlled by light or are light responsive,80 and psilocybin
biosynthesis is also somehow under control of ultraviolet
(UV) radiation. Psilocybe genus mushrooms produce fruit
bodies after UV or blue light irradiation.81 The caps of fruit
bodies formed from poorly enlightened cultures develop blu-
ish phototropic zone originating from oxidation products of
indole compounds. It is also thought that the highest level of
psilocybin coincides with sporulation time.
Analytical Methods Used for
Psilocybin/Psilocin Detection and Isolation
Methods of Extraction
Psilocybin due to the presence of highly polar phos-
phate group is well soluble in water but poorly soluble in
methanol and ethanol. Psilocybin is practically insoluble in
nonpolar solvents such as chloroform and benzene. Psilocin is
slightly more soluble in some nonpolar solvents (1-chlorobutane
or dichloromethane).82–84
Water recrystallized psilocybin (melting point of
220–2808C) forms white needles and contains crystal water.
Psilocybin recrystallized from methanol contains crystal meth-
anol and melts at 185–1958C.85,86
Preparation of fungal material to extraction usually
consists in its air drying at room temperature or slightly elevated
temperature (but not above 408C) and/or freeze-drying.87,88
Comparable analysis reveals that freeze-dried material contains
the highest amounts of psilocybin/psilocin. Pulverization of
dried material is obtained by the use of a mortar or in laboratory
mills and homogenizers.55,74,89,90 Macerated homogenates pre-
pared in a mortar demand separation of solid components.82,91
Extraction of dried and powdered material is usually
assisted by mechanical agitation.74,87,92–94 Sonication in a water
bath was used to assist extraction process.55,95–97 To obtain the
extract of psilocybin/psilocin, a micropercolator was also used.98
The time of extraction, depending on the amount of material,
solvent volume, and the exact mechanical agitation technique,
ranges from a few minutes to several hours.78,88,94 Effectiveness
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Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

of extraction improves and its time may be shortened if the
procedure is carried out at elevated temperature.89,96 However,
heating to the temperature above 708C leads to psilocybin
dephosphorylation, particularly if the extraction is carried out
at acidic pH conditions.82,91 To improve effectiveness, extraction
may be repeated several times and the obtained extracts com-
bined before analysis.89,90,97,98 Methanol is the most often used
solvent for extraction of psilocybin/psilocin because it is able to
denature proteins and to prevent enzymatic conversion of psilo-
cybin to psilocin.55,75,78,87,88,90,92,97 According to Kysilka and
Wurst,94 the optimal solvent for the extraction of psilocybin is
a mixture of methanol and water (75:25) saturated with potas-
sium nitrate. The indole alkaloids could be extracted with 75%
ethanol; however, the obtained solution maintains phosphatase
activity.78,94 It was established that water solutions of acetic acid
are more effective than water solutions of alcohols.78 Ethyl
acetate is also sometimes used for extraction.89 A procedure
of 1-step extraction directly to chloroform was also described.95
Sometimes a method suitable for alkaloids isolation is
used, namely the extraction to acidified water solution followed
by extraction to nonpolar solvent at basic pH.78,82,83,91,99,100 The
macerated material is heated during the procedure what leads to
conversion of psilocybin to psilocin.
Several examples of extraction methods used for
psilocybin/psilocin analysis are briefly described in Table 1.
The obtained extract is separated from suspension by
filtration79,87,92,95,97 or centrifugation.55,82,95
To analyze body fluids (plasma or urine) for the presence
of psilocin, it is necessary to hydrolyze conjugate of psilocin with
glucuronic acid. It is usually performed by the use of Escherichia
coli or Helix pomatia b-glucuronidase at slightly acidic condi-
tions (pH 5–6).102–105 Acidic or alkaline hydrolysis was reported
to be ineffective.104 The addition of methanol to the sample
denatures proteins.104 To deproteinize the ice cold sample,
20% of the solution of polyethylene glycol 6000 was also used.84
Centrifugation is an effective method to separate precipitated
proteins from supernatant containing the exact analytes.104
The obtained supernatant after evaporation to dryness
and dissolving in appropriate solvent is usually directly
analyzed without further treatment. Sometimes the samples
undergo solid-phase extraction for initial purification. For
example, Chem Elut column was used to extract body fluid
samples.55 Initial purification of urine samples containing
psilocin was performed by means of solid-phase extraction
using cation-exchange sorbent.105 To isolate indole alkaloids
from fungal tissue, the crude methanolic extract was purified
by ion-exchange chromatography using cation-exchange
sorbent.75 Purification and preconcentration could be per-
formed using cartridges containing octadecyl sorbent.106
Before high-performance liquid chromatography (HPLC)
analysis, psilocin-containing samples after deproteinization
and alkalization to pH 8.5 were extracted using liquid–liquid
extraction technique or online solid-phase extraction with
cation-exchange sorbent.84
Methods of Qualitative Analysis and Isolation
Blue compound being a product of psilocin oxidation is
easily formed in iodine vapors. Enzymatic oxidation of psilocin
is catalyzed by p-diphenol oxidase.107 Similarly, oxidation with
ferric chloride, a component of the Keller reagent used for Thin
Layer Chromatography (TLC) detection of indole compounds,
makes possible visualization of psilocin spots on TLC plates.51,60
Under UV illumination, psilocin is visible as absorbent
spot.108 Psilocybin shows blue fluorescence under UV light.108
Identification of indole compounds such as psilocybin and psi-
locin in water solutions or after chromatographic separation on
TLC plates may be performed applying the Ehrlich reagent con-
taining p-dimethylbenzaldehyde or para-Dimethylcinnamic acid,
2,4-dimethylcinnamic acid reagent containing 4-(dimethylamino)-
cinnamaldehyde.71,75,108–110 Psilocin was visualized with diazo-
tized p-nitroaniline followed by alkali.105 Psilocin identification
may be based on UV and infrared spectrometry.83
Thin-layer chromatography was reported as an appro-
priate method of psilocybin/psilocin isolation using cellulose

TABLE 1. Exemplary Methods of Extraction Used for Psilocybin/Psilocin Determination

Dry Material/
Solvent Extraction Time Solvent Ratio Extraction Method References
87
Methanol 12 h 250 mg/7 mL Shaking
92
Methanol 30 s 10 mg/2 mL Vortexing
Methanol 30 min — Sonication 55

88
10 mM of HCl in methanol 60 min 100 mg/100 mL Sonication
97
Methanol 15 min 100 mg/3 + 2 mL Sonication
82
10% of acetic acid 0.05 g—0.5 g Homogenization in a mortar
74
Methanol 8h 25–100 mg/10 mL Shaking
69,101
Methanol 12 and 24 h 1g Homogenization in a mortar.
The suspension was left in
methanol for 12 or 24 h
Methanol 3 · 24 h 10 g/3 · 100 mL The suspension was left in 90

methanol for 3 · 24 h
Ethyl acetate 3 · 30 min 20 mg/3 · 1 mL Sonication 89

95
Chloroform 1h 50 mg/1 mL Sonication
99
Water solution of acetic acid, 1 h—maceration, 10 min 10–50 mg/10 mL Maceration, extraction,
pH 4, chloroform chloroform extraction

422
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Ther Drug Monit  Volume 35, Number 4, August 2013
plates or silica gel plates.60,71,87,111–113 The methods of paper
chromatography108 or bidirectional paper chromatography74,75
were also used for separation.
A method of psilocybin isolation using preparative
thin-layer chromatography on silica gel plates followed by
reextraction was developed.90
The method of isolation on cellulose sorbent was also
elaborated followed by crystallization step.90
Analytical Methods of Quantitative Determination
HPLC quantitative analysis of psychoactive substances
originating from fungi of Psilocybe genus is usually performed
by reversed-phase chromatography.84,87,89,92,98,101,102,104,114 Ion-
pair chromatography applying reversed-phase column was also
successfully used.87,98 As a method of determination, ion-
exchange chromatography was also proposed using cation-
exchange sorbent.88 Electrochemical detection is preferred
due to low psilocin absorbance in UV light.84,114 Liquid chro-
matography (LC) combined with mass spectrometry (MS) is an
effective method for detection of psilocybin and psilocin.92,102,104
Thermolabile compounds including psilocybin should
not be analyzed directly by gas chromatography (GC)–MS)
technique. The GC-MS analysis may be performed after deriv-
atization with N-Methyl-N-(trimethylsilyl)trifluoroacetamide
carried out to protect psilocin against thermal decomposi-
tion.95,103,105 Alternatively, electrospray ionization (ESI)–MS
technique89,104,115,116 or chemical-type ionization117 may be used.
Fluorescent psilocin derivative was detected by this ESI-MS
technique.89 It is possible to detect psilocin O-glucuronide
directly without earlier hydrolysis using LC-ESI-MS-MS
technique.104
More sophisticated detection system based on chemilu-
minescence measurements was successfully used for psilocin
determination.69,99
Ion mobility spectrometry may be an alternative
method for rapid psilocin analysis.95
Capillary zone electrophoresis was also used for detection
of psilocybin/psilocin and other indole compounds.55,97,118,119 A
new method was developed for the screening of urine samples for
the presence of 19 drugs of abuse including psilocin.106 The
addition of b-cyclodextrin and organic solvents to the background
electrolyte made possible selective separation of analytes.106
Biologic Activity of Psilocybin/Psilocin in
Mammalian Organisms
Psilocybin/Psilocin Pharmacokinetics
Psilocybin introduced to the digestive tract is very quickly
hydrolyzed. Dephosphorylation under acidic environment of
stomach or under activity of alkaline phosphatase in intestine
TABLE 2. Pharmacokinetic Parameters of Psilocin Determined After Psilocybin Dose Administration
Psilocybin Dose Way of Administration cmax (ng/mL)
0.224 mg/kg (10–20 mg) Orally 8.2
1 mg Intravenous 12.9
0.2 mg/kg Orally 6–21

2013 Lippincott Williams & Wilkins
Psilocin, Ibotenic Acid, and Muscimol
leads to the appearance of active form of the drug, namely
psilocin. Blockage of alkaline phosphatase by means of
competitive substrates prevents the symptoms of intoxication.120
In opposition to psilocybin, psilocin is easily absorbed from the
gastrointestinal tract to the circulating blood.121 Pharmacoki-
netic animal studies on 14C-labeled psilocybin analogue reveal
that psilocybin is absorbed in 50% by the digestive tract after
oral dose application.122,123 Similarly, HPLC studies revealed
about 50% (52.7% 6 20%) bioavailability of psilocin after oral
administration of psilocybin.114 Biodistribution of the drug after
intravenous injection is similar for all organs including brain
(animal study).122 After 8 hours, the tissue content of the drug is
very low except for liver and adrenal tissue still expressing
radioactivity after 48 hours.122
After oral application of psilocybin, plasma concentra-
tion of psilocin increases rapidly, reaches maximum after
50–100 minutes, and slowly decreases during the next 5–
6 hours.84,114,124 Psilocybin injected intravenously rapidly under-
goes dephosphorylation and disappears faster from circulatory
system (when compared with oral administration).114 Pharmaco-
kinetic parameters of psilocin after oral or intravenous adminis-
tration of psilocybin determined in humans are shown in Table 2.
Animal studies reveal that psilocin is in 65% excreted
with urine and in 15%–20% with bile and feces within 8 hours
from administration.122 A small amount (about 10%–20%)
remains in organism for a longer time (14C-labelled metabolites
of psilocin have been identified in urine after 7 days from oral
administration).122 About 25% of the whole dose was shown to
be excreted unaltered in mice.122 Psilocin renal elimination
profiles were studied also in humans.125 The psilocin excretion
rate within 2–4 hours postdrug administration was equal to
55.5 6 33.8 mg/h.125 Within 24 hours, only a small amount
of the whole dose (mean 3.4%) was shown to be excreted as
free psilocin.125 In the same study, it was demonstrated that
after the treatment of urine samples with glucuronidase to mea-
sure the total psilocin concentration, the extent of psilocin
excretion after 24 hours from administration increases only
2-fold (compared with free psilocin measurements). To explain
this, it was proposed that hydroxyl groups of psilocin metab-
olites are also conjugated to O-glucuronide and in this form are
excreted by kidneys.125 The glucuronidation is catalyzed by glu-
curonosyltransferases: microsomal enzymes originating from
liver or intestinal tissues. After oral administration, psilocybin
converted to psilocin is a substrate to UGT1A10 expressed
by small intestine. After absorption within circulatory system
the conversion of psilocin to glucuronide is mediated by
UGT1A9.126 Other identified psilocin metabolites, namely
4-hydroxy-3-yl-acetic acid 4HIAA, 4-hydroxy-3-yl-acetaldehyde
4HIA, and 4-hydroxytryptophol 4I-IT, are the products of its
deamination and oxidation.114,122
tmax (min) t1/2 (min) Fabs (%) References
114
105 163.3 52.7
1.9 74.1 — 114
70–90 135 — 84
423
Stebelska
Psilocin Pharmacodynamics
Psilocin as an alkaloid of the structure resembling
serotonin molecular structure is able to bind and activate
serotonin receptors. Chemical structures of psilocin, its
precursor psilocybin, and serotonin are shown in Figure 1.
Both physiologic and psychotic effects of psilocybin/psilocin
intoxication result from its agonistic activity toward
5-hydroxytryptamine(5-HT) receptors located within or
beyond the central nervous system.
Psilocybin should be considered as a prodrug. Product
of its dephosphorylation, namely psilocin, as relatively
lipophilic substance easily crosses blood–brain barrier.
Psilocybin/psilocin evoked effects are thought to be
mediated mainly by activation of 5-HT2A receptor.127 In vivo
studies in human subjects reveal that ketanserin, 5-HT2A
receptor antagonist, prevents most of the psychotic symptoms
usually developing after psilocybin/psilocin administration.47,128
However, some of the symptoms are not fully diminished and
even intensify under combined psilocybin/ketanserin action
(particularly cognitive deficits measured as difficulties in atten-
tion focusing and “reduction in vigilance”).41,129 The conclusion
of the findings is that serotonergic system mediates psychotic
effects, but they result from both stimulation of 5-HT2A recep-
tors in cortical regions and activation of inhibitory 5-HT1A
receptors located in dorsal raphe nucleus, leading to a decrease
in tonic release of serotonin into prefrontal cortex.
From binding studies, it is known that psilocin as
serotonin agonist binds with high affinity to 5-HT2A (Ki =
6 nM) and 5-HT2C (Ki = 10 nM) and with lower affinity to
5-HT1A receptor (Ki = 190 nM).127,130 Psilocybin and psilocin
are known to inhibit some serotonin biologic activities depen-
dent on 5-HT2C ligation, for example, serotonin-induced pru-
ritus (after its topical administration). Similarly, N-methylated
derivatives of psilocin and psilocybin (see Fig. 2), namely
1-methyl-psilocin and 1-methyl-psilocybin, exhibit comparable
inhibitory action on serotonin activity.131 The 1-methylopsilocin
selectively binds 2 isoforms of 5-HT2C receptor (Ki = 7.0 and
33 nM) and functionally activates the receptor (EC50 = 12 nM).
The experimental data are in agreement with binding energies
calculated from the data originating from computational pre-
diction of the binding site structure in interaction with the stud-
ied derivatives.132 The N-methylated psilocybin (Ki = 540 or
.10,000 nM), similar to psilocybin, inhibits serotonin-induced
scratching. The 1-butyl psilocin (EC50 = 595 nM, Ki = 4.4 or
14 nM) was inactive in the behavioral test. The 4-fluoro-N,
FIGURE 1. Chemical structures of psilocybin, psilocin, and its
endogenous analogue serotonin.
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Ther Drug Monit  Volume 35, Number 4, August 2013
FIGURE 2. Psilocybin/psilocin derivatives tested in papers by
Sard et al131 and Bray et al132
N-dimethyltryptamine (Ki = 82 and 84 nM), despite relatively
poor agonistic activity against 5-HT2C (EC50 = 90 nM), seemed
to be able to inhibit serotonin-induced pruritus.131
However, psilocin psychoactivity is mostly dependent
on ligation of 5-HT2A receptors and inhibitory 5-HT1A receptors.
Although 5-HT2A receptors are expressed by excitatory glu-
tamatergic pyramidal neurons, the overall effect on neuronal
activity is thought to be inhibitory due to the presence of
5-HT2A receptors also on GABAergic interneurons.133
Triggered signaling pathways are different for various
ligands of 5-HT2A receptor depending on ligand-induced con-
formational changes. Normally, serotonin stimulates phospho-
lipase C releasing 2 important signaling molecules inositol
1,4,5-trisphosphate and diacylglycerol and leads to expression
of transcription factor c-fos in a manner dependent on Gq/11
protein.134–136 Subsequent elevation of calcium ions leads to
potassium channels closure and depolarization. Similarly, psilo-
cybin as serotonin agonist by binding to 5-HT2A receptor stim-
ulates hydrolysis of phosphatidylinositol 4,5-bisphosphate.137
Stimulation of 5-HT2A receptor activates also phospholipase
A2 and psilocin, but not its precursor psilocybin, and induces
arachidonic acid release.138 More detailed investigations show
that hallucinogenic serotonin agonists activate additional signal-
ing pathways and induce transcription factors typically after
activation of 5-HT1 receptor, although the receptor is not
involved.136 It is also known that glutamate receptor designated
mGluR2 forms functional complex with 5-HT2A receptor and
modulates the serotonin receptor coupling to Gi protein, what
implicates connection with psychosis induction and explains
why ligation with hallucinogenic molecules results in the acti-
vation of specific signaling pathway.139 Activation of mGluR2
receptor breaks the signaling cascade and corrects symptoms of
psychosis.139
Ex vivo studies on rat brain showed that psilocin affects
hippocampal pyramidal cells. Application of fungal extracts

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Ther Drug Monit  Volume 35, Number 4, August 2013
containing psilocin and psilocybin decreases electrical activity of
these neurons including activity stimulated by glutamic acid.140
The activation of 5-HT1A receptor induces release of Gi/0
protein subunits that activate potassium channel and inactivate
calcium channel leading to hyperpolarization and inhibition of
neuronal activity. Neurons expressing 5-HT1A receptors are
highly responsive to psilocin action. Indeed, direct application
of psilocin to neurons of dorsal raphe nucleus decreases fre-
quency of neuronal firing, what suggests the involvement of
5-HT1A autoreceptor (its activation leads to serotonin decrease
in synaptic cleft).141
According to one of the existing hypotheses, the mech-
anism of waking dream is involved in visual hallucinations
occurrence in course of psilocybin intoxication and in
psychopathology of acute schizophrenic psychosis.142 The state
of waking dream is associated with low level of serotonin
in brain.143–145 During rapid eye movement phase, serotonergic
neurons of dorsal raphe nucleus become inactive and their
normal inhibitory activity on other brain areas transiently dis-
appears. Similar phenomenon takes place under action of psi-
locin possibly due to stimulation of 5-HT1A autoreceptors
present at higher density than other subtypes of 5-HT receptors
on serotonergic neurons of raphe nuclei.146,147
Psilocybin administration decreases binding of radio-
labeled ligand of dopamine D2 receptors in striatum. The
effect results from a rise of endogenic dopamine release and
ligation.48 It should be mentioned here that despite psilocybin
does not show any affinity to dopamine receptor of D2 type,
modulating interactions between serotonergic and dopaminer-
gic neuronal systems are known to exist.
Monoamine oxidase (MAO) inhibitors are taken by
psilocin users to intensify its hallucinogenic effects. MAO is
the enzyme catalyzing serotonin decomposition in synaptic
cleft. Because psilocin may be MAO substrate, its presence
causes inhibition of the enzyme. Brain serotonin is elevated
and simultaneously 5-HIAA level decreases as a result of
MAO blockage.148
Early studies revealed that psilocybin elevates total
level of ATP in central nervous system.149 More detailed
findings applying positron emission tomography determined
elevated glucose metabolic rate under action of psilocybin.
Increased metabolism of some cortical regions (particularly in
anterior cingulate) and decreased metabolic activity in thalamus
were observed after psilocybin administration in humans.43,46
Somatic and Psychotic Effects of Psilocybin/
Psilocin Intoxication
Dose dependent increases of blood pressure and heart
rate were observed in course of psilocybin/psilocin intoxica-
tion.13,15,150–152 The autonomic effects are maximal within
1–2 hours after drug administration.150 Psilocybin elicits neu-
roendocrine effects similar to serotonin-induced changes in
humans150 (see Table 3).
Intensity of psilocybin intoxication depends on a dose of
the drug. Psilocybin at a dose of 8–10 mg (0.1–0.2 mg/kg,
orally) evokes only physiologic symptoms such as mydriasis,
rarely hypotonic reaction associated with nausea and vomiting,
slightly increased blood pressure and accelerated heart fre-
quency, and paresthesias.13,15,124,150–152,156 Pupillary dilation

2013 Lippincott Williams & Wilkins
Psilocin, Ibotenic Acid, and Muscimol
after administration of lower doses only increases sensitiveness
to light, what should not be interpreted as psychotic perceptual
changes.156 However, subjects suffering from headaches are
more sensitive to action of hallucinogens and undergo psy-
chotic syndrome after intake of lower psilocybin doses.157
Administration of higher doses (the amount of about 10–20
mg/70 kg, orally) leads to the elevation of plasma concentra-
tion of the drug up to the value of 5–12 ng/mL.114 Psychotic
syndrome starts to develop when psilocin plasma concentration
reaches the value of about 4–6 ng/ml.114 Psychotic syndrome is
sometimes called schizophrenic phase.146 The symptoms of the
psychosis intensify during the next 30–60 minutes and start to
decline after 2–3 hours.13,50,114,150,151 Psilocybe semilanceata
fruit bodies ingestion results in similar symptoms of intoxica-
tion including psychotic episode.158 It is estimated that the
consumption of hallucinogenic mushrooms in the amount of
2 g (about 10 fruit bodies of P. semilanceata) corresponding to
10–20 mg of psilocybin results in mild psychotic effect usually
experienced as a pleasure (so-called “good trip”). The con-
sumption of 12 g (about 60 specimens) corresponding to
60–120 mg of psilocybin is negatively experienced (as
so-called “bad trip”).159 The psychosis subsides after 5–6 hours
from its appearance. The psychotic symptoms include disor-
ders of visual perception such as illusions or hypnagogic
experiences, alterations of time, color and motion perception
(eg, kaleidoscopic vision), synesthesias, usually positively
experienced sense of derealization and depersonalization,
euphoria sometimes converting to drowsiness, anxiety,
impaired mood, dysphoria, or emotional state described as
extreme fear (mood swings).15,46,48,128,151,153,160–162 Body
image perception is affected—intoxicated subjects reported
impression of swelling and numbness.158 It seems that psi-
locybin impairs cognitive functions: decreases reaction time,
impairs ability to focus and sustain attention mainly by pro-
ducing strong unusual emotions and visions difficult to
ignore, and thinking processes are disturbed by ego disor-
ders and delusions.41–50,128,129 Similar to schizophrenics sub-
jects to whom psilocybin was administered have difficulties
to use contextual information, what may be interpreted as
cognitive deficit.45 However, by lexical decision tasks, it is
possible to show that psilocybin-induced psychosis, similar
to schizophrenia-associated psychosis, predisposes to
semantic network spreading.50 Religious or sensual people
may experience mystical feelings.10–16 Personality character-
ized with specific curiosity (openness to cognitive or per-
ceptual experimentation, also by means of psychoactive
substances) and sensibility to a beauty of nature or works
of art predisposes to pleasurable reactions (including these
of mystical type).163 At high doses, the schizophrenic phase
is followed by the panic attack-like phase because pleasur-
able effects are replaced with dysphoria, paranoid thinking,
and negatively experienced loss of ego control.13,146,158 Low
age and emotional excitability before the drug intake addi-
tionally predispose to such negative responses.163 Accidental
deaths resulted from jumping out of a window after the
consumption of magic mushrooms were reported,8,55 what
suggests unbearable emotional state leading to suicidal
attempt or a state of waking dream (flying is a common
dream motif). The state of psychotic episode obtained after
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Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 3. Dose-Dependent Psychological and Somatic Effects of Psilocybin Treatment

Acute Psychological Effects (at the point of maximal drug effect,
Psilocybin Dose 50–150 min postdrug administration)
Approximations Sensory
of cmax Based Plasma Altered States of Perception, Cognitive
on Data from114 Concentration/cmax Consciousness Hallucinations Mood, Affective States Functions
3–5 mg/70 kg Approximately 2 ng/mL No increases in ASC scores No increase in VR Drowsiness, increased No alternations
(0.045–0.071 mg/kg) score sensitivity
8–10 mg/70 kg Approxmiately 5 ng/mL OB,DED,VR,AA,RV, global Illusions, elementary and
(0.115–0.143 mg/kg) ASC scores [, linearity complex
between a dose and a mean hallucinations,
score or number of synesthesias, sensory
subjects, derealization, alternations, alterations
depersonalization in perception of time
phenomena and space, ASC VR [
15 mg/70 kg Approxmiately 9 ng/mL AMRS scale
(0.215 mg/kg) “general
inactivation” [,
“dreaminess” [,
introversion [
20 mg/70 kg Approximately 14 ng/mL Hallucinogen Rating
(0.315 mg/kg) Scale scores
“somatesthesia,”
“affect,”
“perception,”
“cognition,”
“volition,”
“intensity”[
30 mg/70 kg Approxmiately 19 ng/mL Measurements done based on Mood lability: Not measured 90% of participants
(0.429 mg/kg) questionnaire considering: harmony, reported
sense of unity, happiness, grief, headaches; mean
transcendence of time and joy, anxiety, headache duration
space, sense of sacredness, dysphoria, fear, 16 h
sense of objective reality, paranoid thinking,
positive mood, ineffability, subscale DED[
paradoxicality, transiency;
all ratings [
55
Approximately Femoral venous blood 220 ng/mL Possibly a state of waking Death case Accidental death case,
2 mg/kg, (free psilocin), 4600 ng/mL (total dream (flying is a common death resulted from
90 mg/45–50 kg psilocin); heart blood 60 ng/mL dream motif), euphoria? jumping out of
(free psilocin), 170 ng/mL (total a window after flying
psilocin); postmortem analysis attempt

Psilocybin Dose Subacute Acute Somatic Effects, References, Study

Approximations of cmax Psychological Long-Term Measured Usually 1–2 h Participants,
Based on Data from114 Effects Effects Postdrug Administration Exclusion Criteria
15,47,50,125,150–154
3–5 mg/70 kg Headaches No No changes in cardiac Arterial blood pressure healthy, no personal or
(0.045–0.071 mg/kg) electrophysiology, blood [, heart rate [; rarely family history of psychiatric
pressure, and heart rate tend drop of blood disorders, no history of drug abuse,
to increase pressure with usually had previous experiences
dizziness, fainting, with hallucinogens or cannabis
vomiting
8–10 mg/70 kg Working memory Fatigue Retrospective ratings: positive Arterial blood pressure
(0.115–0.143 mg/kg) impairment headaches change to ASC—56%, in [, heart rate [;
the relationship to the
environment 38%, in
aesthetic experiencing 37%,
in the relationship to one’s
body 30%, in awareness of
personal problems 29%, in
relationships to other 25%;
concentration or memory
problems were reported,
complaints that required
psychotherapy (rarely)

426
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TABLE 3. (Continued ) Dose-Dependent Psychological and Somatic Effects of Psilocybin Treatment

Psilocybin Dose Subacute Acute Somatic Effects, References, Study
Approximations of cmax Psychological Long-Term Measured Usually 1–2 h Participants,
Based on Data from114 Effects Effects Postdrug Administration Exclusion Criteria
15 mg/70 kg Emotional excitation [, Attention Working memory impairment Fatigue, headaches Arterial blood pressure [, heart rate [;
(0.215 mg/kg) dreaminess [, ability Y prolactin [
heightened mood [,
dazed state [,
sensitivity [,
concentration Y,
tiredness [,
introversion [
20 mg/70 kg Emotional excitability [, Impairment of thinking AMRS scale Arterial blood pressure [, heart rate[;
(0.315 mg/kg) anxiety, fear of loss of process, reaction time Y, “dreaminess” [, prolactin [ and out of the normal
self control, paranoid semantic priming promoted melancholia, physiologic range, thyroid-
thinking, labile mood, efficiency-activation stimulating hormone [,
primitive Y, inactivation [, adrenocorticotropic hormone [,
emotionality, introversion [, cortisol [, g-glutamyltransferase [,
emotional blunting extroversion Y, aspartate aminotransferase [
fatigue, headaches
30 mg/70 kg Positive changes in Arterial blood pressure [; 10,11, 13,14, 15, 154

(0.429 mg/kg) mood, personal and, heart rate [ (by about 10 b/ participant were male
social behavior min) Protestant theology
students,
hallucinogen naïve,
double-blind study
(only in theory)
Approximately 2 mg/kg,
90 mg/45–50 kg

ASC, the Altered States of Consciousness Rating Scale 5D consists of the following subscales: oceanic boundlessness (OB; measures: positively experienced phenomena of
derealization, depersonalization, and altered sense of time, elevated mood), dread of ego dissolution (DED; measures negatively sensed depersonalization and derealization, anxiety and
cognitive deficits connected to ego disorders), visionary restructuralization (VR; measures phenomena of elementary and visual (pseudo-)hallucinations, synesthesias, factors
influencing imagination), auditory alterations (AA; measures disorders in auditory perception and hallucinations), reduction of vigilance (RV; measures impairment of alertness and
associated disorders of cognitive functions).155
AMRS, Altman Mania Rating Scale.

even single psilocybin treatment may return particularly
under action of other psychedelic substances such as alcohol
or marihuana (so-called “flashback” effect).8,151,164
Psychological and dose-dependent somatic effects of
psilocybin intoxication are shown in Table 3. Table 4 sum-
marizes pharmacodynamic findings regarding psychotic prop-
erties of psilocybin.
Psilocybin/Psilocin Side Effects and Toxicity
The toxicity of psilocybin is low.165,166 For rodents,
median lethal dose (LD50) equals to 285 mg/kg in mice and
to 280 mg/kg in rats.165 Psilocybin administration results in
transient elevation of blood concentration of some biochemical
markers such as aspartate aminotransferase aspartate transami-
nase and g-glutamyltransferase gamma-glutamyltransferase.150
In humans, even high doses able to induce psychosis
(about 30 mg/70 kg, orally) only slightly and transiently
affect heart rate and blood pressure without noticeable
influence on heart function.13,15,150,152 Arterial pressure increase
partly results from the mechanism controlling circulatory sys-
tem involving central activation of 5-HT2 and 5-HT1A recep-
tors. In addition, psilocin may express ligand activity toward
5-HT2A receptors placed on the surface of smooth muscle
cells leading to vasoconstriction. Disturbances of blood clotting
are possible.167 Rare cardiomyopathies have been reported:
usually single cases of supraventricular tachycardia, myocar-
dial infarction,56 and Takotsubo cardiomyopathy.168 Due to
vasoconstrictor activity of serotonin agonists, the decreased
blood flow through small arteries or blood vessels supplying
nutrition to skin may lead to disturbances of circulation in lower
and upper endings, formation of venous ulcers, and difficulties
in wound healing. The risk of abortion resulted from vaso-
constrictor activity of psilocin as serotonin agonist also exists.
Postdrug administration transient headaches were also
reported.151,154,158 Subchronic intoxication observed during
8 weeks of psilocin daily administration in rats leads to elevation
of magnesium level in plasma, what likely composes protective
mechanism against vasoconstrictor activity of psilocin.166
A destruction of blood–brain barrier under action of psi-
locin is possible.169 Similarly, serotonin released in high
amounts from platelets may damage blood–brain barrier.170
The literature of the subject describes 1 case of 33-year-old
patient who developed a monoparesis of the left leg and bilat-
eral anopia 2 weeks after the consumption of magic mushrooms
due to multifocal cerebral demyelination within the corpus cal-
losum of the right cerebral hemisphere and both optic nerves.169
Potential Therapeutic Psilocybin/
Psilocin Applications
Since the beginning of 1960s of XX century, the
therapeutic potential of psilocybin has been a subject of
scientific interest, particularly in psychiatry and psychotherapy.
Harmfulness of the habit of psilocybin-containing mushrooms
consumption, including all aspects of human health and risk for

2013 Lippincott Williams & Wilkins 427

Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 4. Pharmacodynamic Findings Versus Dose-Dependent Psychological Effects of Psilocybin Treatment

Acute Psychological Effects (at the point of
maximal drug effect, 50–150 min postdrug
Psilocybin Dose cmax administration) Pharmacodynamic Effects
Approximations of cmax
Based on Data From114 Altered States of Consciousness Sensory Perception, Hallucinations Mood, Affective States
0.25 mg/kg + placebo Approxmiately 11 ng/mL Increase in OB, VR and DED scores (about Increase in reaction time [working memory
45% vs placebo conditions) deficits measured with visual manual delayed
response task (DRT)]
0.25 mg/kg+ 20 mg Increases in OB, VR, and DED Reduction of increase in reaction time
ketanserin (5-HT2A scores (about 20% vs placebo
antagonist) conditions)
0.25 mg/kg+ 40 mg No increases in OB, VR, and No increase in reaction time
ketanserin (5-HT2A DED scores (vs placebo
antagonist) conditions)
0.25 mg/kg+ 0.021 mg Increase in OB score (30%), Increase in reaction time
haloperidol (D2 increase in VR scores (40%);
antagonist) increase in DED score (65% vs
placebo conditions)
0.25 mg/kg+ 0.5 mg Increases in OB, VR, and DED Reduction of increase in reaction time
risperidone (mixed 5- scores (about 10%–20% vs
HT2 and D2 placebo conditions)
antagonist)
0.25 mg/kg+ 1 mg No increases in OB, VR, and No increase in reaction time
risperidone (mixed 5- DED scores (vs placebo
HT2 and D2 conditions)
antagonist)
0.25 mg/kg Approxmiately 11 ng/mL Derealization, loss of ego boundaries; OB [, Sensory perception alternations, illusions,
VR [, DED [ complex scenery hallucinations, VR score [
0.215 mg/kg Appr. 9 ng/mL Increases in 5D-ASC scores: OB [ (40%); RV [ (30%); attentional tracking reduced,
DED [ (20%); VR [ (50%); AA [ (15%); spatial working memory not affected
RV [ (30%)
0.215 mg/kg+ 50 mg Increases in 5D-ASC scores: VR [ (15%); RV RV [ (50%); attentional tracking reduced,
ketanserin (5-HT2A [ (50%) spatial working memory not affected
antagonist)
0.115 mg/kg Approxmiately 5 ng/mL Increases in 5D-ASC scores: OB [ (10%); VR Increase in VR score [ (15%). Binocular
[ (15%) rivalry switch rate Y, phase duration [ (from
2 s to about 2.7 s comparing to placebo
conditions, measurement done 90 min after
drug administration)
0.25 mg/kg Approxmiately 11 ng/mL Increases in 5D-ASC scores: OB [ (30%); Increase in VR score [ (40%). Binocular
DED [ (20%); VR [ (40%); AA [ (15%); rivalry switch rate Y, phase duration [ (from
RV [ (25%) 2 s to about 3 s comparing to placebo
conditions, measurement done 90 min after
drug administration)
0.25 mg/kg Approxmiately 11 ng/mL Increases in 5D-ASC scores: OB [(40%); Binocular rivalry switch rate Y, phase duration
DED (20%) [; VR (50%)[; RV(30%) [ [
0.25 mg/kg+ 50 mg Increase in 5D-ASC score RV [ (50%) Binocular rivalry switch rate Y, phase duration
ketanserin (5-HT2A [
antagonist)
0.28 mg/kg Approxmiately 12 ng/mL Brief Psychiatric Rating Scale total score [. Deficits in the AX-Continuous Performance
psychosis factor (conceptual disorganization, Test (measure of visual context-dependent
suspiciousness, hallucinatory behavior, information processing), correct detection of
unusual thought content) [. activation AX sequences Y, incorrect detection of BX
factor (tension, mannerism and posturing, sequences Y
excitement) [. anxiety/depression factor
(somatic concern, anxiety, guilt feelings,
depressive mood) [. anergia factor
(emotional withdrawal, motor retardation,
blunted affect)[. hostility factor (hostility,
suspiciousness, uncooperativeness) [
0.2 mg/kg + activation Approxmiately 9 ng/mL Not measured Difficulties in attention focusing, subjects found
task (word association) the activation task very difficult; number of
associated and spoken words Y

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Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

TABLE 4. (Continued ) Pharmacodynamic Findings Versus Dose-Dependent Psychological Effects of Psilocybin Treatment
Acute Psychological Effects (at the point of
maximal drug effect, 50–150 min postdrug
Psilocybin Dose cmax administration) Pharmacodynamic Effects
Approximations of cmax
Based on Data From114 Altered States of Consciousness Sensory Perception, Hallucinations Mood, Affective States
15 mg for subjects ,50 Approxmiately 19 ng/mL Derealization, depersonalization phenomena, Alternations in the perception of time and
kg, 20 mg for subjects los of ego boundaries 3D-ASC scores [ space, elementary and complex
.50 kg + ketamine + hallucinations, synesthesias
D-amphetamine

Psilocybin Dose References

Approximations of cmax
Based on Data From114 Cognitive Functions
47
0.25 mg/kg + placebo Psilocybin-induced cognitive deficits and
psychotic symptoms are dependent on
activation 5-HT2A receptors
0.25 mg/kg+ 20 mg
ketanserin (5-HT2A
antagonist)
0.25 mg/kg+ 40 mg
ketanserin (5-HT2A
antagonist)
0.25 mg/kg+ 0.021 mg
haloperidol (D2
antagonist)
0.25 mg/kg+ 0.5 mg
risperidone (mixed 5-
HT2 and D2
antagonist)
0.25 mg/kg+ 1 mg
risperidone (mixed 5-
HT2 and D2
antagonist)
48
0.25 mg/kg Emotional disturbances, euphoria, anxiety, 4 Difficulties in thinking, attention Decreased [11C] raclopride receptor binding
subjects had high ratings in OB score, 3 focusing potential bilaterally in the striatum
subjects had high ratings in DED score interpreted as an increase in endogenic
dopamine release and binding
128
0.215 mg/kg Psilocybin-induced activation of 5-HT2A
receptors (leading to increased cortical
stimulation) and activation of 5-HT1A
receptors in raphe nucleus (leading to
a decrease in tonic inhibitory serotonin
release into prefrontal cortex), both effects
impaired attention ability; activation of 5-
HT1A receptors was responsible for the
effect of reduced vigilance
0.215 mg/kg+ 50 mg
ketanserin (5-HT2A
antagonist)
41
0.115 mg/kg Psilocybin-induced activation of 5-HT1A
receptors affects brainstem oscillator
functioning
0.25 mg/kg
129
0.25 mg/kg
0.25 mg/kg+ 50 mg
ketanserin (5-HT2A
antagonist)

(continued on next page )

2013 Lippincott Williams & Wilkins 429

Stebelska
TABLE 4. (Continued ) Pharmacodynamic Findings Versus Dose-Dependent Psychological Effects of Psilocybin Treatment
Psilocybin Dose References
Approximations of cmax
Based on Data From114 Cognitive Functions
0.28 mg/kg Psilocybin psychotic action as 5-HT2A
agonist is associated with context-
dependent information processing, similar
effect was observed in schizophrenics and
in ketamine (NMDA antagonist) treated
subjects, support for the hypothesis of
glutamatergic neurotransmission disruption
as a mechanism underlying psilocybin
effects
0.2 mg/kg + activation Glucose metabolic rates: in right
task (word frontotemporal cortical regions [,
association) particularly in the anterior cingulate [, in
the thalamus Y
15 mg for subjects ,50 Emotional lability, inability to control
kg, 20 mg for subjects emotions, euphoria, dysphoria, paranoid
.50 kg + ketamine + thinking
D-amphetamine
public health and safety, was recently estimated as very
low.8 However, anyone interested in the usage of hallucino-
gens for psychotherapy support should remember that seri-
ously suffering patients would respond poorly to psilocybin
and the drug could further negatively affect their mental
health because it is able to produce schizophrenia mimicking
psychosis.41–50 Psilocybin at high doses (20–30 mg/70 kg)
was shown to induce intense positive mystical-type
experiences in healthy volunteers.10–16 Participants of the
mentioned studies on psilocybin-assisted psychotherapy
self-reported improvements of quality of life after the drug
session.10–16 The results of the psychological experiments
indicate positive influence of psilocybin-induced experien-
ces on quality of social life including mood improvement
and inclination toward more altruistic social behaviors.
However, the acquired data mostly originating from ques-
tionnaire measurements (mystical scale questionnaire, eg)
can be questioned taking into account the known, usually
unpleasant physiologically and emotionally, symptoms of
high-dose psilocybin intoxication. It is surprising that most
of the participants confirmed mood improvement (regarded
as an essential component of mystical experience) under
high-dose psilocybin action. One explanation could be that
they completed questionnaires 7 hours after drug ingestion
(the whole session lasted 8 hours). After that time, all
unpleasant effects of intoxication had already subsided.
The study by Griffiths et al13 was carefully designed to avoid
expectancy effects as much as possible; however, the par-
ticipants were recruited through announcements mentioning
that the planned study aims to assess activity of “psychoac-
tive substance used sacramentally in some cultures.” All
study participants declared positive attitude toward religion
and most of them were members of religious communities.
Postdrug session mood improvements reported by partici-
pants of the experiment may be, at least partly, the effect
430
Ther Drug Monit  Volume 35, Number 4, August 2013
45
43
Difficulties in attention focusing, Glucose metabolic rates: global [ and in 46
thinking process disturbed cortical regions [, subcortical regions [
with derealization and (although less stimulated); The increases
depersonalization phenomena noted in prefrontal cortex, anterior
cingulate, temporomedial cortex and
putamen correlated with psychotic
syndromes, ego pathologies in particular
of personal satisfaction resulted from strong engagement in
absorbing scientific study confirming positive significance
of religion and/or mystical experiences toward those they
were well disposed.
Moreover, the study was designed in such a way that
the participants were constantly monitored during drug session
and also had an opportunity to talk with the observer.
Therefore, when answering mystical scale questionnaire, they
could be prompted by a suggestion, dishonest, or expressed
metaphoric ideas (eg, having in memory and after the
conceptual opposition death/resurrection). Communities con-
glomerated around religious cult are usually oppressive and
their members are trained not only in conformity but also in
cruelty by means of more or less sophisticated dangerous
psychosocial techniques. Therefore, religiously inclined
individuals, particularly if well educated, may mistake,
intentionally or unknowingly, even basic emotions such as
fear and pleasure. Moreover, religiously inclined people
belonging to religious community are instructed to see any
part of their emotional life as something positive and precious
depending on intensity. It may lead to a kind rivalry,
excessive emotional expression or exaltation, progressive
incuriosity (emotional devaluation similar to drug addiction)
and finally result in the discovery of being manipulated or
forced for certain activities by cruel psychosocial or, like in
this case, pharmacologic methods. Such a discovery is never
satisfying (except for conformistic attitude) and may ruin
system of values making impossible social adaptation, what
takes place, for example, in course of schizophrenia. Indeed,
about 40% of participants of the mentioned study experienced
fear or paranoid thinking during drug session.13 Paranoid
thinking included an impression of being manipulated or trea-
ted malevolently—some of the participants decided it is an
occasion to inform about details on their private life.13 Look-
ing for intense, extraordinary emotions, preferably lacking

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Ther Drug Monit  Volume 35, Number 4, August 2013
any connection with reality (escapism) is common to drug
users and alcoholics. Their psychological profile is often
characterized by emotional blunting—in both cases antiso-
cial personality and aggression are more likely to develop in
course of addiction than altruistic attitude. Similarly, as
theatralization of speech and behavior or myth-like confab-
ulations (directly under action of alcohol or due to memory
disorders in course of progressing the Korsakoff syndrome) in
alcoholics, behavioral and mental changes developing under
action of fungal hallucinogens may be directed against others,
particularly if domination becomes the main purpose of the drug
user. Religious context promotes reproduction of the behavioral
pattern. Therefore, expecting positive influence of psilocybin
treatment on quality of social life is simply ingenuousness.
The authors of the mentioned studies remark that fear and para-
noia developing under psilocybin action could be dangerous for
intoxicated subjects.13 Moreover, they take into account possi-
bility of long-term psychological effects of such experiences
and try to measure them. Indeed, personality changes in adults
after psilocybin single (!) treatment were demonstrated: partic-
ularly examined subjects became more open-minded.16 Such
personality change should not be univocally interpreted as pos-
itive—it may reflect serious and dangerous ego disorders.
The therapeutic potential of psilocin and psilocybin was
also tested in therapy of habitual criminals. One study (although
critically reevaluated) demonstrated a short-term therapeutic
effect observed as a decreased frequency of relapse into
crime.171,172 There is also an increasing interest in therapy of
anxiety neurosis, depression, and physical pain in patients with
advanced-stage cancer by the use of psilocybin.36–38 Both these
examples of application are highly controversial and possibly
nonethical.
Psilocybin seemed to be effective in therapy of obses-
sive-compulsive disorder. In standard therapy, selective sero-
tonin reuptake inhibitors are applied, but the results of such
treatment are often unsatisfactory. Single cases supporting the
usefulness of the hallucinogen in correction of obsessive-
compulsive disorder (OCD) symptoms can be found in the
literature.131,173 Moreno and Delgado174 tested effectiveness of
psilocybin in OCD therapy. The study participants have been
delivered 4 different psilocybin doses (the highest dose of
psychedelic action). Therapy was effective for all examined
patients. Similarly, marked effectiveness of psilocybin was
observed in animal models of OCD.131,175,176
Psilocybin, psilocin, or its derivatives may be helpful in
therapy of neurologic or psychiatric diseases associated with
pruritus. Serotonin and 5-HT2C agonists after topical cutaneous
application induce pruritus. In animal studies, the scratching
reaction in response to serotonin subcutaneous injection is
sometimes treated as an animal model of obsessive-compulsive
disorder. The behavioral change is not observed when 5-HT2C
receptors have been blocked. It was established that adminis-
tration of psilocybin or to less extent psilocin diminishes the
itching effect in mice.131
Cluster headache, condition of unknown cause occur-
ring periodically, is often resistant to therapy. Interestingly,
psilocybin treatment results are promising. Examined patients
treated with the drug-reported pain attack interruption and
remission time extension.177

2013 Lippincott Williams & Wilkins
Psilocin, Ibotenic Acid, and Muscimol
IBOTENIC ACID AND MUSCIMOL
Ibotenic Acid and Muscimol as
Secondary Metabolites
Major active substances of Amanita muscaria and
A. pantherina are ibotenic acid and muscimol—the products
of the fungus secondary metabolism, thought to repel insects
and slay their larvae. These 2 species are obligatory ectomy-
corrhizal and grow under deciduous and coniferous trees, espe-
cially beech, birch, and pine, in various types of woods. The
mushrooms are cosmopolitan and occur wild in many conti-
nents (Europe, Africa, Asia, Australia, and America).62,178–180
Muscimol is a product of ibotenic acid decarboxylation.
Both substances are polar and well soluble in water. Bio-
synthetic pathway of ibotenic acid and muscimol includes
conversions of their precursor molecule b-hydroxyglutamic
acid (ring closure and decarboxylation).180
According to some authors, the custom of the cuticule
peeling off before mushrooms consumption aims to avoid
the highest content of these psychoactive substances in the
red cuticle and a yellowish flesh just under it.180 One of the
pigmentary compounds is muscaaurin I (chemical structure
shown in Fig. 3), being an aldimine derivative of ibotenic and
betalamic acids.181,182 Apart from the poisonous properties, to
both invertebrates and vertebrates, the pigment shows antiox-
idative potential and possibly protects against UV radiation.
Quantitative analysis does not support the statement
about higher content of ibotenic acid in the cuticle than in the
flesh. Tsujikawa et al183 examined samples of A. muscaria
and A. pantherina and found that isoxazole compounds tend
to be more concentrated in the flesh than in the red cuticle.
Ibotenic acid contents ranged from 10 to 2845 ppm in the
cap of A. muscaria and from 188 to 269 ppm in the cap of
A. pantherina.183 Muscimol was found in concentrations of
46–1052 ppm in the cap of A. muscaria and 1554–1880 ppm
in the cap of A. pantherina.183 Another analysis of A. musca-
ria fruit bodies184 revealed similar ranges of concentration
(for ibotenic acid, 182–1839 ppm in the cap and 627–1998
ppm in the stem and for muscimol, 46–1203 ppm in the cap
and 82–292 ppm in the stem). Tsunoda et al185 found the
ibotenic content ranging from 258 to 471 ppm and the mus-
cimol content ranging from 18 to 27 ppm in A. muscaria
samples. Størmer et al186 analyzed spores and fresh caps of
fly agarics for the presence of ibotenic acid and they found the
contents to be equal to 0.0054% and 0.0017%, respectively.186
Insecticidal Activity of Ibotenic Acid
The extract of A. muscaria fruit body is thought to be
poisonous or overwhelming for flies. Glutamate and ibotenic
acid are excitatory and inhibitory neurotransmitters in insects
due to the exclusive presence of glutamate-gated chloride
channels in invertebrates.187 Chloride channel activated by
glutamate and ibotenic acid has been also identified in
Drosophila melanogaster.188 By the measurement of hyper-
polarizing response, the presence of an inhibitory glutamate
receptor was demonstrated on the cell body membrane of
a cockroach motor neuron.189 Inhibitory activity of ibotenic
acid and other structurally similar compounds were shown on
snail periodically oscillating neuron.190 Also insect muscle
431
Stebelska
FIGURE 3. Active substances of Amanita muscaria ibotenic,
muscimol, their endogenous analogues glutamic acid and
g-aminobutyric acid (GABA) and a pigmentary component
muscaaurin I.
fibers are responsible for ibotenic acid action. Ibotenic acid
stimulates chloride channels distributed on the nonsynaptic
membrane of the coxal adductor muscle fibers but has no
affinity for glutamate receptors located on the excitatory post-
synaptic membrane.191,192 Inhibitory glutamate-gated chloride
channels have been detected within plasma membranes of
cockroach corpus allatum—densely innervated endocrine
gland synthesizing juvenile hormone. Juvenile hormone con-
trols processes of insect metamorphosis, vitellogenesis, and
reproduction. Ibotenic acid was shown to be able to activate
the channels what resulted in plasma membrane hyperpolar-
ization. Stimulation of these inhibitory receptors is followed
by reduction of juvenile hormone synthesis.193 Stimulation of
excitatory glutamate receptors, which are also present on the
surface of plasma membranes of corpus allatum, results in
antagonistic effect on juvenile hormone production.194,195
Flies of Drosophila genus that normally feed on fly
agaric fruit bodies seem to be resistant to fungal neurotoxins.
Ibotenic acid and to lower extent muscimol are toxic to larvae
of carpophagous species (D. melanogaster and D. immigrans).196
Analytical Methods Used for Ibotenic
Acid/Muscimol Detection and Isolation
Methods of Extraction
Muscimol is a product of ibotenic acid decarboxylation.
Pure ibotenic acid is a crystal colorless substance (melting
point of 150–1528C) readily soluble in water. Similarly, mus-
cimol with the melting point of 1758C, although more hydro-
phobic, is well soluble in water.180 Curiously, pure ibotenic
acid is extremely unstable in solution and rapidly decomposes
to muscimol without involvement of enzymes.197 The addition
of dimethyl sulfoxide to the water solution containing ibotenic
acid reduces its stability. It was established that spontaneous
432
Ther Drug Monit  Volume 35, Number 4, August 2013
decarboxylation of ibotenic acid is significantly accelerated in
a mixture of dimethyl sulfoxide and 3H2O or D2O.197
Ibotenic acid and muscimol were determined in fresh,
frozen, or freeze-dried mushrooms.183–185 The following sol-
vents are usually applied for extraction of ibotenic acid and
muscimol:75% methanol in water or 50%–70% ethanol in
water.183–185,198,199 For briefly described methods of extraction
used for ibotenic acid/muscimol analysis, see also Table 5.
Sometimes extraction to water acidified with formic acid
is applied.200,201 It is thought that decarboxylation of ibotenic
acid to muscimol takes place under these conditions.200 How-
ever, ibotenic acid was detected in water extracts containing
formic acid.186 Methanol or ethanol extracts contain numerous
lipid impurities that are possible to eliminate by several step
extraction with nonpolar solvents.
Parapharmaceuticals, by producer declared to contain
dried A. muscaria fruit bodies, were analyzed for the presence
of ibotenic acid and muscimol applying modified the Stas-
Otto extraction method commonly used for detection of psy-
choactive alkaloids.183 However, it seemed that the method
was not sufficiently sensitive to detect A. muscaria isoxazoles
in the examined material.
Isoxazole derivatives (carbamate in case of muscimol
and carbamate and ethyl ester in case of ibotenic acid) were
extracted to dichloromethane.200 Dansylated and ethyl ester
derivative of ibotenic acid were separated from other compo-
nents of the water–methanol extract by extraction of evapo-
rated residue with ethyl acetate.184
Methods of Qualitative Analysis and Isolation
Qualitative identification of ibotenic acid in water or
water–ethanol extracts of pulverized A. muscaria fruit bodies
is difficult due to the presence of interfering amino acid con-
taminations. Fluorescamine or more often ninhydrin were
used for TLC detection.185,198,202 Detection is easier when
bidirectional TLC is carried out in 2 different solvent sys-
tems.198 Preparative paper chromatography of extracts previ-
ously partly purified using ion-exchange chromatography was
proven to be an effective method of ibotenic acid purification.199
Amino acid contaminants may be eliminated by ion-
exchange chromatography using cation- and anion-exchange
sorbents.185,198–200 Crystallization step is necessary for satis-
factory purification of ibotenic acid.185
Analytical Methods of Quantitative Determination
Reversed-phase chromatography is the most often
used HPLC technique to detect ibotenic acid and/or musci-
mol.185,186,203 Reversed-phase HPLC may be combined with
ion-pair chromatography.186 Dansylated analytes were also
separated using HPLC method on octadecyl column.184 Separa-
tion applying LiChrosorb-NH2 and Nucleosil 5 CN columns was
also effective for muscimol and ibotenic acid determination.204
UV absorption–based detection system was used for
dansylared derivatives quantification.184 For more detailed
identification, eluent from diode detector was analyzed with
mass spectrometer.184 GC–MS technique was used for detec-
tion of carbamate and ethyl ester derivatives of both isoxa-
soles in urine samples.200 Trimethylsilylation at 808C
for 30 minutes with N,O-Bis(trimethylsilyl)trifluoroacetamide

2013 Lippincott Williams & Wilkins
Ther Drug Monit  Volume 35, Number 4, August 2013
TABLE 5. Exemplary Methods of Extraction Used for Ibotenic Acid/Muscimol Determination
Solvent Extraction Time Dry Material/Solvent Ratio mg/mL
75% methanol ? 2.5 g/70 mL
Mixture of methanol: water 7:3 Shaking for 1 min,
sonication for 5 min
50% ethanol 1h
0.1% formic acid in water 10 min Spores: 5 mg/mL, fresh fruit bodies
were suspended with acidified
water (in amount of twice their weight)
results in conversion of isoxazoles to IBO-tri-TMS and MUS-
di-TMS—both are possible to detect applying GC–MS.183
LC/MS/MS technique was used for detection of dansylated
muscimol and dansylated ibotenic acid ethyl ester.184 LC-MS
was also used for direct (without derivatization) detection of
ibotenic acid in water extracts originating from fungal mate-
rial186 and in urine samples.205
Biologic Activity of Ibotenic Acid and
Muscimol in Mammalian Organisms
Ibotenic Acid/Muscimol Pharmacokinetics
A minimal dose to obtain symptoms of central nervous
system intoxication estimated for muscimol is 6 mg. In case
of much less active but more dangerous ibotenic acid, the
amount of 30–60 mg is sufficient for the psychedelic effect.
In contrast to g-aminobutyric acid, muscimol is able to
cross blood–brain barrier possibly by the mechanism of active
transport206–208 or due to muscimol or its metabolite/derivative/
adduct sufficiently high lipid membrane solubility.209 Early
studies resulted in conflicting data regarding muscimol distri-
bution within brain tissue and questioned the a priori accepted
theory of gamma-aminobutyric acid (GABA) receptor popula-
tion as the main binding site of muscimol.209 Muscimol was
shown to be taken up by slices of cortex rat brain and the
uptake was likely dependent on amino acid transport system
localized in nerve terminals.206 The [3H]muscimol binding
studies carried out using crude synaptic membrane preparations
of the rat brain revealed that GABA receptor is the main mus-
cimol binding site and that the binding was in vitro modulated
with melatonin.210 Muscimol high-affinity binding in forebrain
regions such as caudate–putamen, thalamus, and hippocampus
seemed to be dependent on distinct population of GABAA
receptors possibly containing a6 subunit and lacking a1 sub-
unit as shown in knockout study in mice.211
The fast conversion of radiolabeled muscimol to other
metabolites could be inhibited by pretreatment with an
inhibitor of GABA-a-oxoglutarate transaminase.212 It has
been also established with enzyme kinetics studies that mus-
cimol is indeed an amine donor substrate to GABA transam-
inase.201 Another already identified metabolite of the fly
agaric isoxazoles is tricholomic acid shown to be active in
invertebrates as agent able to inhibit neurons.180,190 Tricholo-
mic acid and other unknown ibotenic acid/muscimol metab-
olites may contribute to their activity.209
Because in some cultures drinking urine originating
from intoxicated subjects was practiced for ritual purposes,

2013 Lippincott Williams & Wilkins
Psilocin, Ibotenic Acid, and Muscimol
Extraction Method References
185
Homogenization
50 mg/2 · 2 mL Shaking, sonication 184
198
500–2000 mg/100 mL Grinding and shaking
186
Freezing/thawing, sonication,
homogenization in liquid N2
muscimol was often thought to be mostly unmetabolized.3
Indeed, both active compounds pass through organism
quickly and partly unmetabolized. Ibotenic acid is in vivo
metabolized to muscimol.213 The isoxazoles are excreted by
kidneys. Both ibotenic acid and muscimol were detected in
urine collected 3–8 hours after ingestion of poisonous mush-
rooms in humans (similar concentrations were determined
despite different symptoms and stages of intoxication).200
Ibotenic Acid/Muscimol Pharmacodynamics
Chemical structure of both isoxasoles closely resambles
glutamic acid and a product of its enzymatic decarboxylation,
namely g-aminobutyric acid GABA (see Fig. 3). Structural
similarity of ibotenic acid and muscimol to glutamic acid and
g-aminobutyric acid determinates their ability to bind and
activate receptors of the endogenous neurotransmitters.
Muscimol is an agonist of GABAA and partial agonist
of GABAC receptors but evokes its activity mainly through
GABAA receptors ligation. From radioligand binding study
using bovine brain GABAA receptors reconstituted in lipid
vesicles, it is known that muscimol binds to both high-affinity
(Kd = 10 nM) and low-affinity (Kd = 0.27 mM) sites and func-
tionally activates the receptor (EC50 = 0.2 mM).214 Wild-type rat
GABAA receptor subtype a1b2g2 expressed in cell culture binds
[3H]muscimol with Kd = 21–24 nM.215 Kinetic parameters of
muscimol as GABAA agonist were calculated also from func-
tional assays (apparent affinity KH = 10.9 mM, unbinding rate
koff = 40 /s; microscopic affinity constant Kn = 4.3 nM).216
Behavioral changes induced by muscimol in mice (sedation,
ataxia) depend on its high-affinity binding in cerebral cortex
and hippocampus to a distinct subtype of GABAA receptors
containing a6 subunit and lacking a1 subunit.211
Additionally, muscimol inhibits GABA uptake by neu-
rons and astrocytes and, as already mentioned, is a substrate to
the GABA-metabolizing enzyme, namely GABA transami-
nase.201,212,217,218 Chemical structure of muscimol was used as
a basis for the design of GABA uptake inhibitors and GABA
agonists.218
Ibotenic acid exhibits agonistic activity against N-Methyl-
D-aspartic acid (NMDA) receptors and trans-1-Amino-1,3-
dicarboxycyclopentane receptors (metabotropic quisqualate
Qm receptors). However, neurotoxicity of ibotenic acid
results only from activation of NMDA receptors.219 Activa-
tion of metabotropic Qm receptors followed by cellular signal
cascade initiated by phosphoinositide hydrolysis is not
involved in ibotenic acid–induced excitotoxicity.219 In vitro
experiments revealed that ibotenic acid in contrast to kainic
433
Stebelska
acid does not suppress glutamate ligation and exhibits low
affinity for sites binding radiolabeled kainic acid.220 Therefore,
it is concluded that ibotenic acid is not a ligand of kainate
receptors.
It is postulated that ibotenic acid and muscimol are the
main compounds responsible for hallucinogenic properties of
fly agaric and the mechanism of their toxicity results from
ligation of glutamate and GABA receptors, respectively.
High-dose muscimol treatment potentiates psychosis in
schizophrenics. The subjects to whom muscimol was admin-
istered reported the experience of vivid dreams.221 However,
the studies on the involvement of pedunculopontine tegmen-
tal nucleus in regulation of paradoxical sleep seem to negate
hallucinogenic properties of GABA and glutamate agonists.
Pedunculopontine tegmental nucleus is one of the brain areas
involved in the process of paradoxical sleep appearance and
various paradoxical sleep–associated phenomena. Injections
of glutamic acid and muscimol into pedunculopontine teg-
mental nucleus induce suppression of one of the paradoxical
sleep hallmarks, namely hipoccampal theta rhythm in rats.222
However, it was also shown that muscimol locally adminis-
tered to dorsal raphe nucleus increases REM sleep.223 The
influence of both toxins on sleep architecture is likely the
result of complex GABA and glutamate-dependent mecha-
nisms modulating activity of cholinergic neurons within
pedunculopontine tegmental nucleus and serotonergic neu-
rons of dorsal raphe nucleus.145,201,224
Animal studies demonstrate that due to its inhibitory
properties, muscimol reveals complex neuromodulatory activ-
ity. For example, muscimol (3 mg/kg, intraperitoneally)
evokes serotonin rise and decreases catecholamine levels in
brain.225 Muscimol applied locally was shown to decrease
stratial GABA release in rats.226 The study by Naik et al227
revealed slight increase of the main catecholamine metabolite,
homovanilic acid, in striatum after muscimol administration.
Because muscimol alone decreases motor activity and musci-
mol coadministered with drugs such as cocaine prevents
drug-induced motor hyperactivity dependent on dopamine
neurotransmission, it is assumed that the isoxazole modulates
dopamine release.209
Muscimol, dose dependently, affects encephalogram in
experimental animals distinctly from other typical hallucino-
gens such as lysergic acid diethylamide (LSD) and mesca-
line.228 Particularly, electroencephalogram pattern elicited by
muscimol consists of spikes, what characterizes substances of
convulsant activity.228
The intravenous administration of muscimol reduces
glucose utilization throughout the central nervous system.229,230
However, the pattern of the effect was shown to be different
from the known brain distribution of GABAergic neurons
and GABA receptors.229 Another study revealed correlation
between a decrease of cerebral glucose utilization and a bound
fraction of muscimol (binding dependent on GABAA recep-
tors).231 Decreased brain metabolism was associated with
decreased cerebral blood flow.230 Muscimol lowers cyclic gua-
nosine monophosphate level in cerebellum also in case of its
elevation due to promoting convulsions activity of isoniazid.232
Muscimol administered topically inhibits activity of
spinal neurons in anaesthetized cats and the effect is
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Ther Drug Monit  Volume 35, Number 4, August 2013
diminished with bicuculline (potent GABA antagonist).233
In vitro experiments revealed that activation of GABAA
receptors after ligation of muscimol induces presynaptic
and postsynaptic suppression of synaptic transmission
between muscle afferents and spinal cord motor neurons.234
Muscimol applied microiontophoretically decreased firing
rate of cortical neurons.235
Ibotenic acid is able to excite spinal interneurons and
Renshaw cells as demonstrated in anaesthetized cats.236 The
study reveled that excitatory activity of ibotenic acid is
approximately 8 times higher than glutamate activity. How-
ever, after the recovery of excitation, the neurons became
insensitive to the excitatory agents. The effect was abolished
by bicuculline suggesting involvement of GABAA receptors.
Therefore, it was concluded that ibotenic acid is in vivo
decarboxylated to muscimol.236 Ibotenic acid (16 mg/kg,
intraperitoneally) increases brain monoamines in rodents.225
Somatic and Psychotic Effects Resulted From
Intoxication With A. muscaria and A.a pantherina
First symptoms of fly agaric intoxication apparent within
15–30 minutes after ingestion are signs of muscarinic poisoning
such as nausea, vomiting, diarrhea, vasodilatation, sweating, and
salivation.3,58,207 Then, after about 30 minutes from oral intake,
atropine-like symptoms seem such as mydriasis, xerostomia,
body temperature elevation, slightly increased blood pressure,
drowsiness, amentia, dizziness, hypersensitivity to light, eupho-
ria, motor hyperactivity, hallucinations, and delirium.3,58,59,207
Mystical-type experiences were reported.59,207 The atropine-like
symptoms intensify during the next 2–3 hours. Somnolence
turns into deep sleep—the state is accompanied by skeletal
muscles atony and tendon reflexes debility—the last symptoms
may suggest stroke.207 Similar spectrum of intoxication (hallu-
cinations, muscle twitching, delirium, sleep) has been observed
in human volunteers after pure ibotenic acid and muscimol
administration.237 Poisoning signs decline after about
8 hours.3,58,59,207,237 Somatic and psychotic effects observed in
course of intoxication with poisonous mushrooms or pure mus-
cimol and ibotenic acid are summarized in Table 6.
Within 30 minutes after intravenous injection of crude
extract of A. muscaria changes in biochemical parameters have
been observed in rats including decrease of acetylcholine ester-
ase activity, decline of liver glycogen, decline of blood urea
nitrogen, and increase of blood glucose. No changes of serum
transaminase activity have been observed. After 6 hours, the
parameters come back to the base line.242
Side Effects and Toxicity of Ibotenic Acid/Muscimol
Muscle twitching, spasms, cramps, and even seizures
are frequent symptoms of fly agaric intoxication, particularly
in children.221,237,243 Systemic or local administration of pure
ibotenic acid results in convulsions as established also in
animal studies.244,245
Headaches and fatigue the next day after ingestion were
reported. In case of ibotenic acid intoxication, headache may
be prolonged up to a few weeks.237
Lethal dose LD50 of muscimol determined for rats
is equal to 4.5 mg/kg after intravenous injection and
45 mg/kg after oral administration. The LD50 established

2013 Lippincott Williams & Wilkins
Ther Drug Monit  Volume 35, Number 4, August 2013
TABLE 6. Somatic and Psychotic Symptoms of Intoxication With Amanita muscaria or Amanita pantherina—Mushrooms
Containing Ibotenic Acid and Muscimol. Therapeutic and Side Effects of Muscimol
Dose
Minimal effective dose of muscimol:
6 mg; minimal effective dose of
ibotenic acid: 30–60 mg; one fruit
body of A. muscaria (50–70 g) may
contain up to 70 mg of ibotenic acid
6 fruit bodies of A. muscaria were
eaten by 5 adults
5 caps of A. pantherina were eaten by 2 h after ingestion: nausea,
2 adults
A 72-year-old patient ate a few fruit
bodies of A. pantherina
A. pantherina poisoning case
A. pantherina poisoning case
A. pantherina poisoning case
A. pantherina poisoning case
A. pantherina fruit bodies,
portionconsisting of about one half
cup
A. pantherina fruit bodies,
portionconsisting of about one half
cup
A. pantherina fruit bodies, portion
consisting of about a cup of fried
mushrooms
A. muscaria, 30 g of dried caps

2013 Lippincott Williams & Wilkins
Somatic Symptoms of
Intoxication, Therapeutic, and
Side Effects Psychotic Symptoms of Intoxication
Abdominal pain, nausea
stomach-ache, diarrhea,
vomiting; transiently
increased blood pressure
Diarrhea, vomiting
Vomiting, urine analysis (4 h
postinjection): ibotenic acid
47.4 mg/mL, muscimol
9.9 mg/mL
Normal blood pressure, regular
heartbeat, urine analysis (8 h
postinjection): ibotenic acid
32.2 mg/mL, muscimol
6.0 mg/mL
Nausea, vomiting, normal blood
pressure, regular heartbeat,
urine analysis (3 h
postinjection): ibotenic acid
37.3 mg/mL, muscimol
7.6 mg/mL
Slightly increased blood
pressure, diarrhea, urine
analysis (6 h postinjection):
ibotenic acid 55.2 mg/mL,
muscimol
7.4 mg/mL
No muscle spasms, no vomiting
Nausea
— Dissociative state (lasting 5 h), with no
Muscarinic effects such as
increased salivation and
sweating, slight nausea
Psilocin, Ibotenic Acid, and Muscimol
Long-Term Side
Effects References
58
Visual and auditory hallucinations; 18- Fatigue, stomach Case report
year-old girl lost consciousness complaints the next
day
59
2.5 h after ingestion: mania-like Disordered body Case report
stimulation, agitation, mystical perception,
experiences, 4–5 h after ingestion difficulties to speak
the patient became more anxious, properly
had difficulties to speak properly,
reported vertigo, paresthesias of left
arm
207
Neurologic disorders; hallucinations, Within 10 h after Case report
coma, skeletal muscle flassidity, ingestion neurologic
hyporeflexia, facial assymetry symptoms subsided
202
Hallucinations, the patient had No long-term effects Case report
difficulties in breathing
Dizziness, the patient was conscious No side effects the next
day
Hallucinations No side effect the next
day
Agitation, talkativeness, the patient No side effects the
described the psychotic effects as next day
similar to alcoholic intoxication
3
90 min after ingestion, alternations of No side effects Self
visual perception, wavy motions experimentation
of static objects, altered perception
of size and space, auditory
hallucinations, impaired motor
coordination, pleasurable
emotionally altered state of
consciousness lasting 7 h
Drowsiness, sleep No side effects Case report
No side effects Case report
logical verbal contact with others,
the subject experienced vivid
waking dreams somehow related to
the reality around him
Symptoms developed 1 h after No side effects Self
ingestion, pleasant sedation, experimentation
alternations of visual perception,
impaired motor coordination
(continued on next page )
435
Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 6. (Continued ) Somatic and Psychotic Symptoms of Intoxication With Amanita muscaria or Amanita pantherina—
Mushrooms Containing Ibotenic Acid and Muscimol. Therapeutic and Side Effects of Muscimol
Somatic Symptoms of
Intoxication, Therapeutic, and Long-Term Side
Dose Side Effects Psychotic Symptoms of Intoxication Effects References
Minimal effective dose of muscimol:
6 mg; minimal effective dose of
ibotenic acid: 30–60 mg; one fruit
body of A. muscaria (50–70 g) may
contain up to 70 mg of ibotenic acid
237
Muscimol 5 mg orally No stimulation, inactivation No side effects Self-
experimentation
Muscimol 10 mg orally Myoclonic muscle twitching 90 min postingestion: dizziness, ataxia, No side effects
mood improvement followed by
sleep rich in dreams
Muscimol 15 mg orally Myoclonic muscle cramps, 40 min postingestion: dizziness, Fatigue, inactivation
slightly increased blood reflexes not changed, difficulties in the next day
pressure speaking and attention focusing,
loss of appetite, stimulation, echo-
like pseudohallucinations (visual
and auditory), sleep
Ibotenic acid 20 mg orally Face skin flushing No stimulation, sleep Migraine attack the 237
Self-
(paresthesias), no changes in next day experimentation
blood pressure and heart rate postingestion;
headache (occipitally
localized) for the
next 2 weeks
237
Muscimol 5 mg orally Improvement in concentration Tests in human
performance subjects
Muscimol 10–15 mg orally Impairment in concentration
performance and learning ability
238
Muscimol 5–9 mg orally Improvement in tardive No sedation Schizophrenic
dyskinesia patients
239
Muscimol 7 mg orally Improvement in tardive Impairment in attention performance Schizophrenic
dykinesia and saccadic patients
distractibility
240,241
Muscimol orally. Within 120 min from ingestion: - Patients
prolactin [, growth hormone with the
[, dose-dependent increases Huntington
disease and
chronic
schizophrenia
221
Muscimol 5 mg orally. - No relief of psychosis resulted from Schizophrenic
a disease, sedation, relaxation patients
Muscimol 7–10 mg orally. Myoclonic muscle twitching Increases in psychosis symptoms
measurements: confusion, affect,
thought disorder, maximal effect
1–2 h after administration, sleep,
vivid dreams

in mice are 2.5 mg/kg (intraperitoneally), 3.8 mg/kg (sub-
cutaneously), and 5.62 mg/kg (i.v.). The LD50 determined
in rabbits is 10 mg/kg (orally). Lethal doses of ibotenic acid
established in rats are 128 mg/kg (orally) and 42 mg/kg
(intravenously). Ibotenic acid LD50 determined in mice
equals to 15 mg/kg (intravenoulsy) and 38 mg/kg
(orally).246
To investigate an involvement of a particular brain area
in the studied brain activity, ibotenic acid as a cytotoxic agent
is directly injected into the brain tissue in vivo (animal
experiments).219,247–249 Due to its cytotoxic properties, ibo-
tenic acid is used to model the Alzheimer disease in rats. This
compound injected into the entorhinal cortex causes neuronal
losses via hippocampal perforant pathway resembling defects
observed in the Alzheimer disease.250
Less toxic muscimol applied in similar experiments
pharmacologically mimics cytotoxic agents by transient
inactivation of neuronal activity. The mechanism of its action
involves activation of GABA receptors followed by neuronal
depolarization caused by chloride ions influx. GABAA and
GABAC receptors are GABA-gated ion channels specific
for chloride ions. After ligation, they trigger inward chlo-
ride current, what inhibits neuronal activity.251–253 For
example, it was established that microinjected muscimol
inhibits the dorsomedial hypothalamus and the caudal region
of the lateral/dorsolateral periaqueductal gray. The inactivation

436
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Ther Drug Monit  Volume 35, Number 4, August 2013
was able to diminish autonomic responses (such as increases in
heart rate and blood pressure, adrenocorticotropic hormone
elevation) to stress conditions in rats.254
Potential Therapeutic Muscimol Applications
Due to its inhibitory activity, muscimol possesses anal-
gesic properties. When injected intravenously, muscimol poten-
tiates analgesic effect of pilocarpine in rats227 and morphine in
rats and mice.255,256 All these effects are mediated by GABAer-
gic system. Intrathecally injected muscimol, by GABAA ago-
nistic activity, blocks nociception mediated by excitatory
aminoacids.257 Muscimol was shown to potentiate anaesthetic
activity of propofol, what allowed to establish GABAergic
transmission involvement in the mechanism of anaesthesia
induced by propofol.258
Intrathalamic microinjections of muscimol were able to
reduce tremor in patients suffering from essential tremor, what
would implicate therapeutic use of the isoxazole compound to
treat the neurologic disorder.258
Muscimol applied systemically or topically to cortex
was shown to block or delay drug-induced convulsions in
animals.232,260,261 Pretreatment with muscimol (transmenin-
geal administration) at 2.5 mM concentration prevents
acetylcholine-induced seizures in rats and monkeys without
serious side effects.261 Therefore, it could be potentially used
for epilepsy treatment.
Moreover, despite psychoactive properties, muscimol
applied in relatively low doses improves schizophrenia
symptoms working as an anxiolytic likely by the mechanisms
of neuronal activity suppression and/or paradoxical sleep
suppression.221 In agreement with the finding of muscimol
anxiolytic activity are results originating from later studies
on the activation of GABA receptors localized in the lateral
nucleus of the amygdale. Muscimol injections depending on
concentration and the presence of selective antagonists affect
fear learning and memory in rats. Particularly, activation of
GABAA receptors by relatively low doses of muscimol in
the presence of GABAC antagonist leads to fear learning
blockade.262 Sedative effect of muscimol was also shown in
mice.212,237
Muscimol was proven to improve tardive dyskinesia in
schizophrenic patients.238,239
Muscimol is able to prevent cellular death in ischaemia–
reperfusion conditions when released in high amounts gluta-
mate exhibits cytotoxic activity. The preventive activity of
muscimol was demonstrated in animal model of ischaemia–
reperfusion.263,264 Muscimol stimulation of modulating hetero-
receptor GABAA on glutamatergic neurons reduces activation of
glutamate receptor NMDA, what protects pyramidal neurons
against excessive excitability.265 The precise mechanisms of
muscimol protective action is not known—it possibly involves
inhibition of NMDA-dependent activation of neuronal nitric
oxide synthase nNOS.266 In vitro studies revealed that GABA
and muscimol inhibit NO synthesis in rat ileal nerve terminals
through activation of GABAA and GABAC receptors.267 How-
ever, muscimol independently on its GABAA agonistic activity
was also shown to stimulate NO release.268 Antiinflammatory
activity of muscimol in endotoxemia was recently demonstrated
in mice.268,269

2013 Lippincott Williams & Wilkins
Psilocin, Ibotenic Acid, and Muscimol
CONCLUDING REMARKS
Psilocybin/psilocin referring scientific literature, including
analytical chemistry studies, is quite rich. Therapeutic strategies
based on psilocybin biologic activity are being developed.
Agonists of serotonin receptors may find application in
therapeutic areas such as psychiatry and neurology.4 The drugs
of activity against serotonergic system such as ergotamine and
sumatriptan are already used to treat migraine headaches.270,271
The results of search for potent and selective agonists of 5-HT2C
receptors among psilocybin derivatives seem to be promising.
On the other hand, psilocybin/psilocin–induced psychosis is
commonly used to model schizophrenia, what allows for better
understanding of its complex etiology and psychopathology.
Hallucinogens of fly agaric seem to be far less frequently
a subject of chemical analysis and studies on the mechanisms of
their biologic activity in humans. It is not surprising if one is
aware of the use of A. muscaria isoxazole neurotoxins for a long
time in neurobiology where ibotenic acid commonly serves as
brain-lesioning agent. Less poisonous muscimol and its deriva-
tives possess high therapeutic potential in neurology and psy-
chiatry, although the potential is still insufficiently put to good
account.
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