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From: (H-Man)
Subject:  mdma article #9
Message-ID: <>
Date: Sat, 3 Jul 1993 17:52:36 GMT

                            JAMA(R) 1988; 260: 51-55
                                  July 1, 1988
LENGTH: 3242 words
TITLE: (+/-) 3, 4-Methylenedioxymethamphetamine Selectively Damages Central
Serotonergic Neurons in Nonhuman Primates
AUTHOR: George A. Ricaurte, MD, PhD; Lysia S. Forno, MD; Mary A. Wilson; Louis
E. DeLanney, PhD; Ian Irwin; Mark E. Molliver, MD; J. William Langston, MD
ED/SECT: Thomas P. Stossel, MD, Section Editor
ABSTRACT: (+/-) 3, 4-Methylenedioxymethamphetamine ( MDMA)  is a popular
recreational drug that has been proposed to be useful as an adjunct to
psychotherapy.  This study assessed the neurotoxic potential of  MDMA  in
nonhuman primates.  Monkeys were repeatedly administered doses (2.50, 3.75, and
5.00 mg/kg) of  MDMA  subcutaneously and analyzed for regional brain content of
serotonin and 5-hydroxyindoleacetic acid two weeks later.  In all regions of
the monkey brain examined, MDMA produced a selective dose-related depletion
of serotonin and 5-hydroxyindoleacetic acid.  These neurochemical deficits
were associated with evidence of structural damage to serotonergic nerve
fibers.  In addition, MDMA produced pathological changes in nerve cell
bodies in the dorsal, but not median, raphe nucleus.  These results indicate
that MDMA is a selective serotonergic neurotoxin in nonhuman primates and
that humans using this drug may be at risk for incurring central
serotonergic neuronal damage. 
   RECREATIONAL abuse of controlled substance analogues ("designer drugs")
potentially poses a major health problem. [n1-n3] (+/-) 3,
4-Methylenedioxymethamphetamine ( MDMA) , variously known on the street as
" Ecstasy, " "Adam," or "XTC," [n4] is an analogue of the controlled substance
(+/-) 3, 4-methylenedioxyamphetamine (MDA).  Presently,  MDMA  is one of the
more popular recreational drugs in the United States. [n5] It has been
estimated that 30 000 capsules of the drug are sold each month (R. K.
Siegel, PhD, unpublished data, 1985).  It has also been proposed that MDMA
may be useful as an adjunct to insight-oriented psychotherapy. [n6, n7] This
suggestion is based largely on subjective reports that MDMA improves
interpersonal communication and enhances emotional awareness.
   In 1985, the Drug Enforcement Agency placed  MDMA  on Schedule I of
controlled substances, citing increasing recreational use of this drug and
expressing concern that  MDMA  might cause neurological damage. [n8] This
concern arose largely because of evidence that MDA (the N-desmethyl derivative
of  MDMA)  destroys central serotonergic nerve terminals in rats. [n9] Recent
studies indicate that MDMA, like MDA, is toxic to serotonergic nerve
terminals in the rodent brain. [n10-n15] However, findings in rats appear to
have done little to deter recreational use of MDMA.  At least in part, this
may be because studies in rodents do not always accurately predict drug
toxicity in humans.  For example, 1-methyl-4-phenyl-1, 2, 3,
6-tetrahydropyridine (MPTP) is relatively inactive in rats [n16, n17] but
profoundly toxic in primates. [n18, n19] Conversely, 1, 2, 3,
6-tetrahydro-1-methyl-4-(methylpyrrol-2-yl)pyridine, an analogue of MPTP, is
very toxic in rodents [n20] but inactive orally in primates. [n21] In
addition, differences in the way rodents and primates metabolize
amphetamines [n22] may alter the neurotoxic effects of these drugs. For
these reasons, we thought it critical to assess the neurotoxic activity of
MDMA in nonhuman primates. 
   Seventeen monkeys were used in this study.  Eleven female squirrel monkeys
(Saimiri sciureus) 6 to 8 years of age and weighing 0.6 to 0.7 kg were used for
neurochemical studies and for anatomic studies of the raphe nuclei.  Three
female rhesus monkeys (Macaca mulatta) 1.5 to 4.0 years of age and weighing 2.5
to 3.5 kg and two female and one male cynomolgus monkeys (Macaca fascicularis)
weighing 2.0 to 4.5 kg were used for immunohistochemical studies.  No
differences in response to  MDMA  were noted among the three species.
Drug Treatment
   The hydrochloride salt of  MDMA  was administered subcutaneously twice daily
at 0800 and 1700 hours for four consecutive days.  This dosing regimen was used
to permit comparison of the present results with those previously obtained in
rodents. [n12, n14] For neurochemical studies, eight of 11 squirrel monkeys
were administered the following doses of MDMA according to the
above-mentioned schedule of drug administration: 2.50 mg/kg (n = 2), 3.75
mg/kg (n = 3), and 5.00 mg/kg (n = 3).  The three remaining squirrel monkeys
served as untreated controls.  For immunohistochemical studies, three of six
macaque monkeys were given the high-dose (5.00 mg/kg) regimen of MDMA; the
other three untreated monkeys served as controls.
   Two weeks after drug treatment, the monkeys were killed under deep ether
anesthesia.  The brain was removed from the skull, and the brainstem was
dissected away and placed in 10% formol saline for later anatomical study.  The
forebrain was dissected over ice, and the various brain regions were isolated
for analysis of monoamine content.  Concentrations of serotonin,
5-hydroxyindoleacetic acid, dopamine, and norepinephrine were measured by
reverse-phase high-performance liquid chromatography coupled with
electro-chemical detection, using the method of Kotake et al [n23] with minor
modification. [n24]
   For routine histological studies of the raphe nuclei, the brainstems of
three monkeys that had received the 5-mg/kg regimen of MDMA two weeks
previously were immersion-fixed in 10% formol saline for one week prior to
paraffin embedding and staining.  Sections were stained with
hematoxylin-eosin, Luxol fast blue (LFB)-cresyl violet, LFB-periodic
acid-Schiff (PAS), or LFB-Bielschowsky.  For immunohistochemical studies of
serotonergic nerve fibers in the forebrain, three monkeys that had received
the 5-mg/kg regimen of MDMA two weeks previously and three controls were
administered the monoamine oxidase inhibitor trans-2-phenylcyclopropylamine
(10 mg/kg intraperitoneally) one hour prior to being killed by intracardiac
perfusion under deep sodium pentobarbital anesthesia.  After the vascular
tree was cleared with ice-cold phosphate-buffered saline, perfusion was
continued with 4% paraformaldehyde, pH 6.5, followed by 4% paraformaldehyde
and 0.12% glutaraldehyde (pH 9.5).  Tissue blocks were placed in buffered 4%
paraformaldehyde for seven hours and then in 10% dimethyl sulfoxide in
phosphate-buffered saline overnight.  Frozen sections (30 mum) were
incubated in an antiserotonin antisera (R8) diluted 1:5000 (or in
anti-tyrosine hydroxylase antisera diluted 1 U:48 mL) in phosphate-buffered 
saline with 0.2% octyl phenoxy polyethoxyethanol (Triton X-100) and 1% normal
goat serum at 4 degrees C for three days.  The antibody was visualized with a
peroxidase-labeled avidin-biotin complex (Vector Laboratories Inc, Burlingame,
Calif), and staining was enhanced with the osmiophilic reaction sequence of
Gerfen. [n25]
   After a simple one-way analysis of variance showed an F value of P<.05,
individual values were compared with the control using a two-tailed Student's t
test.  Thereafter, regression analysis was performed and the 3df between groups
were partitioned into a regression component (1 df) and a deviation from
regression component (2df).
   Dopamine hydrochloride, norepinephrine hydrochloride, and serotonin
creatinine sulfate were purchased from the Sigma Chemical Company, St Louis;
MDMA hydrochloride was provided by David Nichols, PhD, Department of Medicinal
Chemistry, Purdue University, Lafayette, Ind, and the National Institute of
Drug Abuse.  Tranylcypromine (tranyl-2-phenylcyclopropylamine) was purchased
from Regis Chemical Company, Morton Grove, Ill.  The rabbit antiserotonin was
generated by H. Lidov against serotonin conjugated to bovine serum albumin with
formaldehyde.  Rabbit anti-tyrosine hydroxylase antisera was purchased from
Eugene Tech International Inc, Allendale, NJ.
   Dose Response. -- Measurement of serotonin two weeks after drug treatment
showed that multiple subcutaneous doses of  MDMA  92.50, 3.75, and 5.00 mg/kg)
produced a dose-related depletion of serotonin in the somatosensory cortex of
the monkey, with the lowest dose (2.50 mg/kg) producing a 44% depletion and the
highest dose (5.00 mg/kg) producing a 90% depletion (Table 1).  Statistical
analysis (simple analysis of variance followed linear regression with
partitioning of the degrees of freedom into a regression component [1 df] and a
deviation from regression component [2 df] revealed that linearity explained
virtually all of the variability between doses (r = .97).  The deviation from
regression component was not statistically significant (F [2, 8] = 2.28;
Table 1. -- Dose-Related Decrease in Serotonin Concentration in the
Somatosensory Cortex of the Monkey Two Weeks After Administration of  MDMA
   Regional Effects. -- Multiple doses of  MDMA  also produced large depletions
of serotonin in the caudate nucleus, putamen, hippocampus, hypothalamus, and
thalamus of the monkey (Table 2).  One of the most severely affected areas was
the cerebral cortex (Table 2), where the lowest dose (2.5 mg/kg) of  MDMA
produced a 44% depletion of serotonin (Table 1).
Table 2. -- Regional Concentrations of Serotonin in the Monkey Brain Two Weeks
After Administration of  MDMA  (5 mg/kg)
   Other Markers. -- Measurement of 5-hydroxyindoleacetic acid, another
chemical marker for serotonergic nerve fibers, showed that multiple doses of
MDMA also markedly reduced the concentration of this compound (Table 3).
Concentrations of 5-hydroxyindoleacetic acid were reduced by 84% in the
neocortex, 76% in the caudate nucleus, 75% in the hippocampus, and 40% in
the hypothalamus. 
Table 3. -- Decreased Concentration of 5HIAA in the Monkey Brain Two Weeks
After Administration of  MDMA  (5 mg/kg)
   Selectivity. -- Measurement of dopamine and norepinephrine concentrations in
monkeys receiving the highest dose (5 mg/kg) showed that  MDMA  produced no
depletion of dopamine or norepinephrine (Table 4).
Table 4. -- Unchanged Concentrations of Dopamine and Norepinephrine in the
Monkey Brain Two Weeks After Administration of  MDMA  (5 mg/kg)
   Nerve Fibers. -- Immunohistochemical studies performed to assess the
structural integrity of serotonergic nerve fiber projections to the forebrain
demonstrated a marked reduction in the number and density of
serotoninimmunoreactive axons throughout the cerebral cortex of three of three
monkeys receiving the 5-mg/kg dose of  MDMA  (Fig 1).  In addition, at higher
power, some serotonergic axons appeared swollen and misshapen.  Staining
with an antibody to tyrosine hydrosylase revealed no evidence of damage to
catecholamine-containing nerve fibers in the cerebral cortex.
   Cell Bodies. -- Examination of nerve cell bodies in the raphe nuclei of the
monkeys receiving the highest dose of  MDMA  (5 mg/kg) showed that while  MDMA
produced no obvious cell loss in either the dorsal or median raphe nuclei, the
drug induced striking cytopathological changes in nerve cells of the dorsal
raphe nucleus.  In three of three of these animals, hematoxylineosin-stained
paraffin sections of the dorsal raphe nucleus showed numerous, somewhat
shrunken nerve cells that contained brownish-red spherical cytoplasmic
inclusions that displaced the nucleus to the periphery of the cell (Fig 2,
top left).  In LFB-PAS-stained sections, the inclusions appeared granular
and were vividly PAS positive (Fig 2, bottom right).  This staining reaction
suggests the presence of an increased amount of ceroid or lipofuscin,
possibly due to lipid peroxidation of cell components and subsequent
phagolysosomal activity.  The presence of lipofuscin within the inclusions
was confirmed by a number of staining procedures.  Specifically, the
granules were autofluorescent in ultraviolet light, acid fast in
Ziehl-Nielsen stain for lipofuscin, and positive with e chmorl's reaction
and Sudan Black B stain.  Glycogen did not account for the staining, as
demonstrated in PAS stain with and without diastase. 
   No abnormal inclusion-bearing cells were found in the median raphe nucleus,
in other raphe nuclei, or in nonserotonergic nuclei such as the substantia
nigra or locus ceruleus.  No similar inclusions were found in ten control
monkeys of varying ages (including three 15- to 20year-old monkeys),
although some increased lipofuscin pigment was occasionally found in the
older animals. (Seven of these ten animals were not formally part of the
present study but had served as controls in other experiments.  The brains
of these seven animals were fixed by immersion in 10% formol saline.)
   The major finding of this study is that central serotonergic neurons in
nonhuman primates are highly vulnerable to toxic effects of  MDMA.   Compared
with the rodent, [n10-n15] the primate has been found to be approximately four
to eight times more sensitive.  In the monkey, a dose of 2.5 mg/kg produces a
44% depletion of serotonin in the cerebral cortex (Table 1).  By contrast, in
the rat a 10- to 20-mg/kg dose is required to produce a comparable effect.
[n14] Also of note is the fact that in the primate small increments in dose
from 2.50 mg/kg to 3.75 and 5.00 mg/kg produced 78% and 90% depletions of
serotonin, respectively (Table 1).  This indicates that the dose-response
curve of MDMA in the monkey is steep, suggesting that the margin of safety
of MDMA in humans may be narrow.
   The striking loss of serotonin-immunoreactive nerve fibers in the cerebral
cortex of the  MDMA -treated primate (Fig 1) suggests that  MDMA  produces a
long-term depletion of serotonin by actually damaging serotonergic nerve
fibers. Axonal damage is further suggested by the swollen and distorted
appearance of some of the remaining fibers.  Morphological evidence of nerve
fiber damage is important because it suggests that the prolonged depletion
of serotonin induced by MDMA is not merely due to a pharmacologic action of
the drug, but rather represents a neurotoxic effect.  Anatomical studies in
rats have led to a similar conclusion. [n12, n14]
   It is not yet known whether the effects of  MDMA  on serotonergic neurons in
the primate are permanent or reversible.  Under some circumstances,
regeneration of serotonergic nerve fibers in the central nervous system can
take place. [n26] However, for axon regrowth to occur, the cell body must be
preserved.  It remains to be determined if serotonin-containing cell bodies
in the dorsal raphe nucleus of the MDMA -treated primate survive beyond two
weeks.  If they do, and if regeneration of nerve fibers takes place, it is
still not certain that the new fibers would establish normal connections.
For functional integrity to be maintained, normal connections would need to
be reestablished.  It will be important to determine if this occurs in MDMA
-treated animals.
   This study provides the first direct evidence that serotonergic cell bodies,
as well as nerve fibers, are affected by  MDMA.   As shown in Fig 2, the
pathological change in cell bodies involves formation of intracytoplasmic
inclusions.  These inclusions resemble the more eosinophilic but usually
PAS-negative inclusions recently described in monkeys given MPTP, [n27] a
compound that destroys nigral cell bodies. [n18, n19] Whether the inclusions
in the  MDMA -treated primate herald nerve cell death or reflect a metabolic
response of the cell body to anoxal injury is not yet known but needs to be
ascertained because, if cell-body death occurs, the possibility of axonal
regeneration would be precluded.
   The fact that abnormal inclusions were found in nerve cells of the dorsal,
but not median, raphe nucleus is noteworthy because it suggests that  MDMA
selectively damages a particular subset of serotonergic neurons in the brain
(ie, the B7 group of Dahlstrom and Fuxe).  That this is the case is also
suggested by the recent finding in the rat that serotonergic nerve fibers
arising from the dorsal, but not median, raphe nucleus are damaged by  MDMA.
[n12, n28] Taken together, these findings indicate that MDMA is likely to be
a valuable new tool for further defining the functional anatomy of different 
serotonergic cell groups in the mammalian brain.
   The mechanism by which  MDMA  exerts its toxic effects on central
serotonergic neurons is at present not well understood.  Like a number of other
ring-substituted amphetamines (eg, p-chloroamphetamine, fenfluramine
hydrochloride, MDA),  MDMA  appears to release serotonin. [n29-n31] Commins and
colleagues [n32] have proposed that  MDMA  and related compounds destroy
serotonergic neurons by releasing large amounts of serotonin and inducing
endogenous formation of 5, 6-dihydroxytryptamine, a well-known serotonergic
neurotoxin. [n33] However, other investigators [n34] maintain that the
degenerative effects of ring-substituted amphetamines may be mediated by a
toxic metabolite.  It remains to be determined which, if either, of these
possibilities proves correct.
   The results of this study raise concern that humans presently using  MDMA
may be incurring serotonergic neuronal damage.  The fact that monkeys are
considerably more sensitive than rats to the toxic effects of  MDMA  suggests
that humans may be even more sensitive.  Before extrapolating the present
results to humans, however, it should be noted that monkeys were given multiple
rather than single doses of  MDMA  and that the drug was given subcutaneously
rather than orally. Humans generally take  MDMA  via the oral route and use
single 1.7- to 2.7-mg/kg doses of the drug, usually weeks apart, although some
individuals have used higher and more frequent doses. [n4] It remains to be
determined if administration of MDMA to monkeys in a pattern identical to
that used by humans produces similar neurotoxicity.  In this regard,
however, it is important to bear in mind that the sensitivity of human and
nonhuman primates to the toxic effects of MDMA may not be the same.  In
fact, humans are generally regarded as being more sensitive than monkeys to
the toxic effects of drugs. For example, humans are fivefold to tenfold more
sensitive than monkeys to the toxic effects of MPTP (compare references 19
and 35).  In view of these considerations, it would seem prudent for humans
to exercise caution in the use of MDMA.  Caution may also be warranted in
the use of fenfluramine, a ring-substituted amphetamine that is closely
related to MDMA and is currently prescribed for obesity [n36] and autism.
   From an experimental standpoint,  MDMA  appears to hold promise as a
systemically active toxin that can be used to study the functional consequences
of altered serotonergic neurotransmission in higher animals.  Clinically, it
will be important to determine if humans who have taken  MDMA  show biochemical
signs of serotonergic neurotoxicity (eg, decreased 5-hydroxyindoleacetic acid
concentration in their cerebrospinal fluid).  If they do, it will be
critical to ascertain if these individuals have any functional impairment.
In particular, such individuals will need to be evaluated for possible
disorders of sleep, mood, sexual function, appetite regulation, or pain
perception, since central serotonergic neurons have been implicated in all
of these functions. [n38, n39] These studies could offer the unique
opportunity to better delineate the neurobiology of central serotonergic
neurons in the human brain, something that until now has not been possible.
SUPPLEMENTARY INFORMATION: From the Departments of Neurology and Neuroscience,
The Johns Hopkins University School of Medicine, Baltimore (Drs Ricaurte and
Molliver and Ms Wilson); the Department of Pathology, Veterans Administration
Medical Center, Palo Alto, Calif (Dr Forno); and the institute for Medical
Research, San Jose, Calif (Drs Ricaurte, DeLanney, and Langston and Mr Irwin).
   Reprint requests to the Department of Neurology, Francis Scott Key Medical
Center, The Johns Hopkins Health Center, 4940 Eastern Ave, Baltimore, MD 21224
(Dr Ricaurte).
   This work was supported in part by the Multidisciplinary Association for
Psychedelic Studies, Sarasota, Fla; the Veterans Administration Medical
Research Program; ational Institutes of Health grant NS21011 (M.E.M.); and
California Public Health Foundation Ltd subcontract 091A-701. One of the
authors (M.A.W.) was supported by the L. P. Markey Fund.
   We thank Lorrene Davis-Ritchie, ZoAnn McBride, David Rosner, and Patrice
Carr for expert technical assistance.
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GRAPHIC: Figure 1, Serotonin-immunoreactive fibers in somatosensory cortex
(area 3) of cynomolgus monkey. Serotonergic axons form dense terminal plexus in
control animal, in methylenedioxymethamphetamine ( MDMA) -treated animal (5
mg/kg), there is marked decrease in density of serotonergic axons after a
two-week survival period. Changes in somatosensory cortex are representative of
serotonergic denervation caused by MDMA throughout cerebral cortex. Scale
bar, 100 mum; Figure 2, Nerve cells in dorsal raphe nucleus of
methylenedioxymethamphetamine ( MDMA) -treated squirrel monkey. Several of
slightly shrunken nerve cells contain intracytoplasmic inclusion
(hematoxylin-eosin, x 550). Nerve cells in dorsal raphe nucleus from untreated
11-year-old squirrel monkey, (hematoxylin-eosin, x 550). Close-up view of
one of abnormal inclusion-bearing cells in dorsal raphe nucleus of the MDMA
-treated squirrel monkey (hematoxylin-eosin, oil immersion, x 1480).
Close-up view of nerve cells in dorsal raphe nucleus to show vividly
periodic acid-Schiff-positive granular inclusions in perikarya of several
nerve cells (Luxol fast blue-periodic acid-Schiff stain, oil immersion, x