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Science, Vol 297, Issue 5590, 2260-2263 , 27 September
2002
[DOI:
10.1126/science.1074501]
Severe
Dopaminergic Neurotoxicity in Primates After a Common Recreational Dose
Regimen of MDMA ("Ecstasy")
George A. Ricaurte,1*
Jie Yuan,1 George
Hatzidimitriou,1 Branden J.
Cord,2 Una D. McCann3
The prevailing view is that the popular recreational
drug(±)3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy") is
aselective serotonin neurotoxin in animals and possibly in
humans.Nonhuman primates exposed to several sequential doses of
MDMA, aregimen modeled after one used by humans, developed
severe braindopaminergic neurotoxicity, in addition to less
pronounced serotonergicneurotoxicity. MDMA neurotoxicity was
associated with increasedvulnerability to motor dysfunction
secondary to dopamine depletion.These results have implications
for mechanisms of MDMA neurotoxicityand suggest that
recreational MDMA users may unwittingly be puttingthemselves at
risk, either as young adults or later in life, fordeveloping neuropsychiatric
disorders related to brain dopamine and/orserotonin deficiency.
1
Department of Neurology,
2 Department of Neurosciences,
3 Department of Psychiatry, Johns Hopkins
BayviewMedical Center, Johns Hopkins University School of Medicine,
Baltimore,MD 21224, USA.
* To whom correspondence should
be addressed. E-mail:Ricaurte{at}jhmi.edu
MDMA ("ecstasy") has become apopular
recreational drug internationally (1, 2).In
the 1980s, MDMA was generally used on college campuses, with
mostindividuals taking no more than one or two 75- to
150-mg doses, about1.6 to 2.4 mg per kilogram of body
weight (mg/kg), twice monthly(3). More recently, MDMA
is increasingly used in the contextof large, all-night dance parties
where partygoers regard the drug assafe and consume multiple doses
during the night (4,5).
MDMA appears to carry risks beyond the sociobehavioral effectsassociated
with drug abuse. Experimental animals treated with MDMA
showevidence of brain serotonin neurotoxicity (6-8),
and MDMA-induced serotonin neurotoxicity may also occur in humans
(9, 10). Virtually all animal
species tested until now show long-term effects on brain
serotonin neurons but nolasting effects on either brain dopamine
or norepinephrine (NE) neurons(6-8). In the
mouse, dopamine neurons are affected, but serotonin neurons are
spared (11, 12).
We used nonhuman primates to evaluate the neurotoxic potential of adose
regimen modeled closely after one often used by MDMA users
atall-night dance parties. Squirrel monkeys (Saimiri sciureus) were
given MDMA at a dosage of 2 mg/kg, three times, at 3-hour intervals,
for a total dose of 6 mg/kg (13). Of five monkeystreated
with MDMA, three tolerated drug treatment without any
apparentdifficulty. One monkey became less mobile and had an
unstable,tentative gait after the second dose, and therefore it
was not giventhe third planned dose. The fifth monkey developed malignanthyperthermia
and died within hours of receiving the last dose of MDMA.Two
weeks after MDMA treatment, the three monkeys that tolerated
drugtreatment were examined for chemical and anatomic markers of
brainserotonin neurons (13), along with three
saline-treatedcontrol animals. These studies revealed lasting reductions
in regionalbrain serotonin, serotonin's major metabolite (5-hydroxyindoleaceticacid,
or 5-HIAA), and the serotonin transporter (SERT).
Anatomicstudies (13) supported these observations, showingreductions
in the density of serotonin- and SERT-immunoreactive (SERT-IR)
axons in some cortical regions (Fig. 1). Six weeksafter
MDMA treatment, the monkey that received only two doses of
MDMAwas evaluated and found to also have long-lasting reductions
inserotonin axonal markers; serotonin, 5-HIAA, and SERT in the
caudatenucleus of this animal were reduced by 37, 48, and
40%, respectively.
 Fig. 1.Effect
of MDMA on regional brain(A) serotonin (5-HT), (B) 5-HIAA,
and(C) SERT in squirrel monkeys 2 weeks after drug
treatment.Results shown represent the mean ± SEM (n = 3 animals
per group). DPM, disintegrations per minute; Fc,frontal cortex; Pc,
parietal cortex; Tc, temporal cortex; Oc, occipitalcortex; Hc, hippocampus;
Cd, caudate nucleus; Put, putamen. Asteriskdesignates P < 0.05, determined
by individualcomparison to control after one-way analysis of variance
showed anF value with P < 0.05. (D)5-HT-
and (E) SERT-IR axons in the parietal cortex of acontrol monkey
(left) and a monkey treated with MDMA 2 weeks previously(right).
Dark-field photomicrographs of the coronal plane are shown;scale
bar = 100 µm. (F) radioisotope[3H]RTI-55-labeled
SERT in coronal section of a controlmonkey (CON) and a monkey treated with
MDMA 2 weeks previously. Thescale on the right shows the density of
binding sites designated bycolor expressed in nanocuries (nCi) per mg of
tissue.[View Larger
Version of this Image (0K GIF file)]
These same monkeys had marked reductions in various
markers of striataldopaminergic axons (Fig. 2). The
profound loss of striataldopaminergic axonal markers was
consistently observed in all monkeysexamined, including the
animal that received only two MDMA doses;dopamine,
3,4-dihydroxyphenylacetic acid (DOPAC), and the
dopaminetransporter (DAT) in the caudate nucleus of this animal
were reduced by65, 77, and 51%, respectively, 6 weeks
after MDMA exposure. The lossof dopaminergic axonal markers was
greater than the loss ofserotonergic axonal markers. Morphologic studies
revealed correspondingreductions in the density of striatal DAT-
and tyrosine hydroxylase(TH)-IR axons throughout the striatal complex,
with somesparing of the more caudal portion of the caudate nucleus
(Fig. 2).Quantitative autoradiography studies (13) confirmed thesevere reductions in striatal DAT density
(Fig. 2).
 Fig. 2.Effect
of MDMA treatment on striatalconcentrations of (A) dopamine (DA), (B)[3H]WIN35,428-labeledDAT,
(C) DOPAC, and (D) radioisotope[3H]MTBZ-labeled
vesicular monoamine transporter-2(VMAT) in squirrel monkeys examined
2 weeks after MDMAtreatment. (E)[3H]RTI-121-labeled
DAT in coronalsection of a control monkey and a monkey treated with MDMA
2 weekspreviously. The scale on the right shows the density of
bindingsites designated by color expressed in nCi/mg of tissue. (Fand
G) DAT-IR axons and axon terminals in the striatum of(F) a control
monkey and (G) a monkey treated with MDMA 2 weekspreviously. (H
and I) TH-IR axons and axonterminals in the striatum of (H) a
control monkey and (I) a monkeytreated with MDMA 2 weeks previously.
Dark-field photomicrographs ofthe sagittal plane are shown; scale
bar = 100 µm.[View Larger Version of this
Image (0K GIF file)]
To determine whether the severe long-lasting decrements
in dopaminergicaxonal markers in squirrel monkeys were unique to this
primate species,we tested the effects of the same MDMA regimen in
baboons (Papioanubis) (13). Again, one of five
animals died, thistime shortly after receiving only two doses of
MDMA. Malignanthyperthermia (up to 41.6oC) was again
an important factor.A second baboon appeared unstable after the
second dose of MDMA andtherefore received only two of the three
planned doses. Two to 8 weeksafter treatment, the four
surviving MDMA-treated baboons, along withthree saline-treated control
animals, underwent chemical and anatomicstudies of brain dopamine
and serotonin neurons (13).Neurochemical and quantitative
autoradiography studies again revealed aprofound loss of
striatal dopaminergic axonal markers (Fig.3). Dopaminergic
deficits in the striatum of the baboon thatreceived only two
MDMA doses were as severe as those in the baboonsthat received
all three doses. Baboons also developed less severe, butsignificant,
long-term reductions in regional brain serotonergic neuronal
markers (Fig. 3).
 Fig. 3.Effect
of MDMA treatment on striatalconcentrations of (A) dopamine, (B)[3H]WIN35,428-labeled
DAT, (C) DOPAC, and(D) [3H]MTBZ-labeled VMAT in
baboons examined2 weeks after MDMA treatment. (E)[3H]RTI-121-labeled
DAT in a coronal section of acontrol baboon and a baboon treated with MDMA
2 weeks previously. Thescale on the right shows the density of binding
sites designated bycolor expressed in nCi/mg of tissue. (F)
Serotonin(5-HT), (G) 5-HIAA, and (H) SERT inbaboons
2 weeks after MDMA treatment. (I)[3H]RTI-55-labeled
SERT in a coronal section of acontrol baboon and a baboon treated with MDMA
2 weeks previously.The scale on the right shows the density of binding
sites designated bycolor expressed in nCi/mg of tissue.[View Larger Version of this
Image (0K GIF file)]
To evaluate the selectivity of the observed effects, we
assessed thestatus of noradrenergic neurons in both monkeys and baboons.
MDMAproduced no long-term effects on NE levels or the density of
NEtransporters in the brain of either primate species (figs. S1
and S2).Consistent with the lack of a long-term effect of MDMA
on theconcentrations of NE and its transporter, the density of
TH-IR axons inthe cerebral cortex of MDMA-treated monkeys was
unaffected (fig. S1).
To determine that the lasting loss of chemical and anatomic markers
ofstriatal dopaminergic and serotonergic axons and axon terminals
was, infact, due to a neurotoxic insult rather than to lingering
acutepharmacological effects of MDMA, we used Fink and Heimer's
method(14), which allows for selective silver
impregnationof degenerating axons and axon terminals. A monkey
treated with MDMAand evaluated 31/2 days later (13)
had denseargyrophilic debris characteristic of axon terminal degeneration
in thestriatum (Fig. 4). No such degenerative debris
was evident inthe striatum of the control animal. We also found
a vigorous glialresponse (Fig. 4) in adjacent striatal
tissue sections processed forglial fibrillary acidic protein
(GFAP) immunocytochemistry(13).
 Fig. 4.Silver-stained
coronal sections through thecaudate nucleus of (A) a control monkey
and (B) amonkey treated with MDMA (one dose of 2 mg/kg at
3-hour intervals,three times) 31/2 days previously. Fine argyrophilic
debris inthe MDMA-treated monkey is characteristic of axon
terminaldegeneration, as demonstrated by the Fink-Heimer method(14). Scale bar = 10 µm. (C) Paucity
ofGFAP-IR cells in the caudate nucleus of a control monkey and(D)
marked increase in the number of GFAP-IR cells in thestriatum of a monkey
treated with MDMA 31/2 days previously.Scale bar = 10 µm.[View Larger Version of this
Image (0K GIF file)]
We next explored the possibility that monkeys with
MDMA-induceddopaminergic neurotoxicity (with no evidence of Parkinsonism)
are atincreased risk for the development of motor dysfunction secondary
todopamine depletion (13). Monkeys (n = 3)
received a challenge dose regimen of alpha-methyl-para-tyrosine (AMPT)1 week
before and 1 week after MDMA treatment. Using a dosage
regimenof AMPT that gradually reduces brain dopamine concentrations, we
hopedto model the progressive decline in brain dopaminergic function
thatoccurs with normal aging (15). Compared to
their baseline,monkeys were more sensitive to AMPT-induced motor
dysfunction 1 weekafter MDMA treatment (fig. S3).
We report severe, functionally significant dopaminergic
neurotoxicity,along with more modest serotonergic neurotoxicity, in
primates treatedwith doses of MDMA modeled after those commonly used
by recreationalMDMA users. Earlier studies in nonhuman primates have
generallyinvolved administration of higher MDMA doses (5 or
10 mg/kg) twicedaily (morning and evening) for 4 consecutive
days. These dosageregimens typically engendered more severe but highly
selectivetoxicity toward brain serotonin neurons, with no
long-term effects onbrain dopamine neurons (16-18).
Because the drugregimens used in previous studies did not model those
used by most MDMAusers, the possibility remained that occasional MDMA
users might not beat risk for neurotoxic injury. The present results,
however, indicatethat even individuals who use MDMA on one
occasion may be at risk forsubstantial brain injury if they use
two or three sequential doses,hours apart, as is often the case
in recreational settings.
In the present studies, MDMA was given by a systemic
route(subcutaneously in squirrel monkeys and intramuscularly in baboons),
whereas humans generally take MDMA orally. It is possible that humansare
at a decreased risk for neurotoxic injury because of differences
inthe route of administration. However, in the case of MDMA,
oraladministration offers little or no significant neuroprotection (19-22). Even if some degree of protection wereafforded
by oral administration, the profound loss of dopaminergic neuronal
markers seen in both primate species suggests that significant neurotoxicity
would still occur. Moreover, individual doses of MDMAused in
this study are lower than those typically used by humans
(1.6 to 2.4 mg/kg), once adjusted with interspecies dose scaling
methods(23). Hence, any protection that might be
associated withoral administration would likely be offset by
increasing the dose ofMDMA used in this study to the human equivalent.
It is not uncommon forrecreational MDMA users to use repeated
doses of the drug on more thanone occasion or more than two or
three repeated doses per session.
The present findings challenge the commonly held notion that MDMAis a
selective brain serotonin neurotoxin and carry important publichealth
and scientific implications. Based on MDMA use pattern, theremay
be two separate MDMA cohorts: those with selective brain serotonergic
neurotoxicity and those with combined serotonergic andmore
severe dopaminergic neural injury. It will be exceedingly important
to consider this when attempting to identify and interpret functional
consequences of MDMA use in humans. Cognitive abnormalities identified
in MDMA users (24-26) may be related, at
least in part, to dopaminergic rather than serotonergic neurotoxicity.
The present findings also have implications for effortsaimed at
identifying the mechanisms of MDMA neurotoxicity. Previousstudies
have identified a metabolite of MDMA that might be
responsiblefor its neurotoxic effects, the 6-hydroxydopamine analog2-(methylamino)-1-(2,4,5-trihydroxyphenyl)
propane(27-29). Because this toxic
metaboliteinduced both dopaminergic and serotonergic neurotoxicity,
and becauseMDMA was believed to be a selective serotonin
neurotoxin, it receivedlittle further attention. This 6-hydroxydopamine
analog of MDMAobviously warrants closer scrutiny as a potential
mediator of MDMAneurotoxicity.
The development of profound dopaminergic neurotoxicity after two orthree
sequential MDMA doses of 2 mg/kg each leads one to question
whatdistinguishes this particular drug regimen from the 4-day,
twice daily,higher-dose regimen that engenders selective serotonergic
neurotoxicity(16-22). One possibility is that
the nonlinearpharmacokinetic profile of MDMA, such as that
demonstrated in humans inthe setting of closely spaced repeated
dosing (30,31), leads to
prolonged elevated brain levels of MDMA (orits metabolites) and
that protracted exposure to MDMA renders dopamineneurons vulnerable
to its toxic effects. An alternative (although not mutually
exclusive) explanation is that repeated closely spaced dosesof
MDMA lead to higher elevations in body temperature, which is
knownto augment MDMA neurotoxicity (32). Additional
studies areneeded to evaluate these possibilities, in addition to
alternativehypotheses.
In light of the present findings, and given the fact that MDMA use
iswidespread and increasing, one might ask why more cases of
MDMA-inducedParkinsonism (33) have not been reported.
There are multiplepotential explanations, but only two will be mentioned.
First,Parkinsonism does not generally become clinically apparent
until morethan 70 to 80% of brain dopamine has been depleted.
Therefore,substantial MDMA-induced dopaminergic neurotoxicity could
occur yetremain occult until unmasked by other processes (such
as drug-inducedinterference with dopaminergic neurotransmission or
decline in braindopamine with advancing age). Second, until now,
the potential for MDMAto damage brain dopamine neurons in primates
has not been appreciatedand, therefore, MDMA neurotoxicity has
not been considered in thedifferential diagnosis of Parkinsonism in
young adults. It is possiblethat some of the more recent cases of
suspected young-onsetParkinson's disease might be related to
MDMA exposure but that thislink has not been recognized.
These findings suggest that humans who use repeated doses of MDMA
overseveral hours are at high risk for incurring severe brain
dopaminergicneural injury (along with significant serotonergic neurotoxicity).
Thisinjury, together with the decline in dopaminergic function
known tooccur with age (15), may put these individuals
at increasedrisk for developing Parkinsonism and other
neuropsychiatric diseasesinvolving brain dopamine/serotonin deficiency,
either as young adultsor later in life.
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Supported by USPHS grants DA5707, DA 13790, DA 09487, DA
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29 May 2002; accepted 14 August2002
10.1126/science.1074501
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Volume 297, Number 5590,
Issue of 27 Sep 2002, pp. 2260-2263.
Copyright © 2002 by The American Association for the
Advancement of Science. All rights reserved.
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