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3.7 Suministro de heroína por prescripción médica

3,4-Methylenedioxymethamphetamine
and 3,4
Methylenedioxyamphetamine Destroy Serotonin Terminals in Rat Brain: Quantification of
Neurodegeneration By Measurement of
[³H]Paroxetine-Labeled
Serotonin Uptake Sites‘

GEORGE BATTAGLIA, S. Y. YEH, ELIZABETH
O’HEARN, MARK E. MOLLIVER, MICHAEL J. KUHAR and ERROL B. DE SOUZA
Neuroscience Branch (G.B., S.Y.Y.,
M.J.K., E.B.D.S.), Addiction Research Center, National institute on Drug Abuse, Baltimore,
Maryland and
Department of Neuroscience (E.O’H., M.E.M.), Johns Hopkins University, School of Medicine,
Baltimore, Maryland
Accepted for publication May
15,
1987

ABSTRACT
This study examines the effects of repeated systemic adminis-tion (20 mg/kg s.c., twice
daily for 4 days) of 3,4-methylene.xymethamphetamine (MDMA) and
3,4methylenedioxyamphetamine (MDA) on levels of brain monoamines, their metabolites and on
the density of monoamine uptake sites in various regions of rat brain. Marked reductions
(30-60%) in the concentration of 5-hydroxyindoleacetic acid were observed in cerebral
cortex, hippocampus, striaturn, hypothalamus and midbrain at 2 weeks after a 4-day
treatment regimen of MDMA or MDA; less consistent reductions in serotonin (5-HT) content
were observed in these brain regions. In addition, both MDMA and MDA caused comparable and
substantial reductions (50-75%) in the Pensity of [‚Hlparoxetine-labeled 5-HT uptake sites
in all brainregions
examined. In
contrast, neither MDMA nor MDA caused any widespread or long-term changes in the content
of the catechotaminergic markers (i.e., norepinephrine, dopamine, 3,4
dihydroxyphenylacetic acid and homovanillic acid) or in the number of [3
HImazindol-labeled norepinephrine or dopamine uptake sites in the brain regions examined.
These data demonstrate that MDMA and MDA cause long-lasting neurotoxic effects with
respect to both the functional and structural integrity of serotonergic neurons in brain.
Furthermore, our measurement of reductions in the density of 5-HT uptake sites provides a
means for quantification of the neurodegenerative effects of MDMA and MDA on presynaptic
5-HT terminals.

MDMA and MDA
are ring-substituted derivatives of methamphetamine and amphetamine, respectively. These
compounds have been reported to exhibit both stimulatory and pychotomimetic properties
(Anderson et al., 1978; Braun et al., ‚1980). Structural similarities
between MDMA, MDA and their respective parent compounds suggest that these amphetaminelike
compounds may exert effects on monoaminergic systems in brain comparable to those of the
amphetamines. Various regimens of amphetamine and methamphetamine administration have been
reported to significantly alter both DA and 5HT neurotransmission in brain in various
species including rat, guinea pig and monkey (Hotchkiss and Gibb, 1980; Wagner et al.,
1979, 1980; Ellison et al., 1978; Lorenz, 1981; Ricaurte et al., 1980;
Sciden et al., 1975/76). The effects that have been observed include decreases in
monoamine and monoamine metabolite levels, synthetic enzyme activities and decreases in
the maximal activity of neuronal uptake systems.

In contrast to the reported effects of the amphetamines on both
dopaminergic and serotonergic neurons, recent evidence suggests that their methylenedioxy
derivatives, MDMA and MDA, may cause preferential long-terin alterations of serotonergic
parameters in brain similar to those observed for the halogenated amphetamines (Fuller,
1978). Marked reductions in 5-HT content and in the maximal activity of 5-HT uptake
systems have been observed in discrete regions of rat brain up to 14 days after repeated
systemic administration of MDA (Ricaurte et al., 1985). In addition, both acute and
short-term administration of MDMA or MDA have been shown to produce reductions in the
content of 5-HT and 5-HIAA and decreases in tryptophan hydroxylase activity in various
brain regions (Schmidt et al., 1986; Stone et al., 1986). Although these
findings indicate that MDMA and MDA can significantly alter erotonergic transmission in
brain, it is unclear whether the reductions in various parameters of serotonergic function

(i.e., 5-11T and 5-HIAA content, tryptophan hydroxylase and
5-HT uptake activity) are the consequence of decreased functional activity in otherwise
structurally intact neurons or are subsequent to the destruction of 5HT axons and/or
terminals. Therefore, although MDMA and MDA may be considered neurotoxic with respect to
their effects on 5-HT metabolic processes, additional measures are necessary to
substantiate the neurodegenerative effects of these drugs on 5-HT neurons.

The present study was designed to assess and quantify the neurotoxic
and neurodegenerative effects of short-term administration of MDMA and MDA on monoamine
neurons in various regions of rat brain. Inasmuch as 5-11T uptake sites are highly
concentrated on 5-HT-containing nerve terminals (Kuhar and Aghajanian, 1973), we can
directly quantify the degree of n eu rodege ne ration of serotonergic terminals by
measuring the reductions in uptake site density. We have reported recently the feasibility
of using radioligand binding techniques and high affinity ligands; for 5-HT, DA and NE
uptake sites to monitor the loss of monoamine terminals (Yeh

et al., 1986). We report here that MDMA and MDA cause marked reductions in
various markers (i.e., 5-HT, 5-HIAA and 5-HT uptake sites) of 5-HT terminals.
Marked reductions in the density of 5-HT uptake sites in all brain regions examined which
reflect the loss of 5HT axons and/or terminals indicate that both MDMA and MDA are potent,
long-lasting and widely acting 5-HT neurotoxins.

Materials and
Methods

Studies in
this report were carried out in accordance with the Declaration of Helsinki and/or with
the Guide for the Care and Use of Laboratory Animals as adopted and promulgated li~ the
National Institutes of Health.

Materials.
[‚H]Mazindol (19.7 Ci/mmol), [‚Hjparoxetine (22.7 Ci/ mmol) and Formula 963 scintillation
fluid were obtained from New England Nuclear (Boston, MA). MDA and MDMA were provided by
the National Institute on Drug Abuse. Mazindol was supplied by Sandoz Pharmaceuticals
(East Hanover, NJ) and citalopram was obtained from Lundbeck (Copenhagen, Denmark). All
other compounds were obtained from Sigma Chemical Co. (St. Louis, MO).

 

Animal treatments. Male Sprague-Dawley rats (Harlan Industries, Indianapolis,
IN), weighing 175 to 200 g, were housed under standard laboratory conditions (12-hr
light/dark cycle) and had access to food and water ad libitum. Rats were injected s.c.
twice a day, (approximately every 12 hr) with 20 mg/kg of MDA or MDMA (expressed as the
free base) or saline vehicle (I ml/kg) for 4 consecutive days and then sacrificed 2 weeks
after the last injection. This treatment regimen has been shown to produce maximal changes
in brain 5-HT content after administration of MDA (Ricaurte et al., 1985). Cerebral cortex
(i.e., frontoparietal cortex), hippocampus, striatum, hypothalamus and midbrain were
dissected as described previously (Glowinski and Iverson, 1966), frozen immediately in
liquid nitrogen then stored at -70*C until required for assay (less than 2 months).

HPLC determinations. Tissues from various brain regions were
homogenized with 0A N HCIO, containing 0.1% cysteine. Catecholamines, 5-HT and their
metabolites were measured with electrochemical detection after separation by
reversed-phase HPLC. Samples were chromat.ographed on a Ci,, radial-pak cartridge and
elated with a mobile phase at a flow rate of 0.9 ml/min. Each liter of the mobile phase
contained 0.1 g of EDTA, 1.3 g of sodium heptyl sulfonate, 8 ml of triethylamineand 45 ml
of acetonitrile and the p1l was adjusted to 2.85 with phosphoric acid (4.5 nil).
Concentrations of catecholamines, 5HT and their metabolites in the brain tissues were
calculated from standard curves, using DHBA as an int(mial sLandard. The standard curves
were linear from 3 to 100 ng/ml (60-2000 pg/20 pl). The retention times for NE, DHBA, DA,
DOPAC, 5-HIAA, HVA and 5HT were 3.15, 4.5, 6.46, 8.25, 13.52, 16.08 and 17.45 inin,
respectively.

 

Measurement of uptake sites. Frozen brain regions front individual
saline- and drug-treated rats were weighed and prepared as described previously (Habert et
at., 1985). Tissues were placed in 50 volumes of ice-cold 50 mM Tris-HCI (pH 7A), 120 mM
NaCl, 5 inNI KCI and homogenized using a Brinkman polytron (setting of 5, 2

X 30 se,c). The homogenate was centrifigued at 48,000 x g for 10 min with a
subsequent resuspension and wash of the pellet. The final pellet was resuspended in the
same buffer to a concentration of 15 mg of wet wt./
MI.

NE and DA
uptake sites were measured using 6 nM [‚Hlinazindol according to the method of Javitch et
al. (1984). NE uptake site specific binding was defined as the difference between total
binding and that in the presence of 0.3 uM DMI. DA uptake site specific binding was
determined as the difference between the binding of [‚111mazindol in the presence of
0.3,uM DMI and that in the presence of I uM mazindol. DMI at a concentration of 0.3 pM has
been shown previously to selectively preclude the binding of [‚H]mazindol to NE uptake
sites without affecting binding to DA uptake sites. Assays were carried out in 0.5 ml of
50 mM Tris-HCI, 120 mM NaCl and 5 mM KC1 (pH 7.9 at 4’C). Tubes containing drugs and
tissue (5-8 mg of wet wt./tube) were incubated on ice for 60 min then filtered rapidly
over Whatman GF/C filters which had been presoaked in 0.05% PEI and washed with 15 ml of
cold buffer.

[³H]Paroxetine binding to 5-HT uptake sites was carried out in the assay buffer
described above with the following modifications of the original protocol (Habert et at.,
1985). Increasing concentrations of I’Hjparoxetine (0.002-0.25 nM) in the absence and
presence of I ‚UM citalopram were
incubated with 1.5 mg of tissue in 5 ml of buffer. Tubes were incubated for 2 hr at 22*C
in order to ensure that the lowest concentrations of ligand had reached equilibrium. All
tubes were then rapidly filtered over PEIpresoaked GF/C filters and washed with 15 ml of
buffer. Filters were then equilibrated with 5 ml of Formula 963 scintillation fluid and
counted at 54% efficiency in a Beckman 3801 scintillation spectrometer. For each
concentration of [‚Hlparoxetine, a parallel set of tubes was prepared with tissue and
incubated similar to the experimental tubes. The tubes were centrifuged and an aliquot of
the supernatant was taken in order to determine the „true“ free 1’HJ paroxetine
concentrations. Due to the high affinity (Kn = 10-20 pM) of this radioligand and
consequent significant depletion which occurred at the lower concentrations, it was
necessary to measure the actual amount of [‚H]paroxetine at each of the concentration
points. Proteins were determined according to the method of Lowry et al. (1951).

Data analyses. The data from the radioligand binding studies were analyzed by
the computer program „EBDA“ (McPherson, 1983) which provides initial estimates
of equilibrium binding parameters by Scatchard and Hill analyses. All data were analyzed
for statistical significance using one-way analysis of variance and Duncan’s multiple
range test.

Results

071.gif (36381 bytes)
F
ig 1 Effect of repeated
systemic administration of MDMA and MDA on the concentration of (A) 5-HT and (B) its
metabolite 5-HIAA in various brain regions. Rats were injected s.c. twice daily for 4 days
with drug (20 mg/kg) or saline vehicle (1 mllkg) and sacrificed at 2 weeks after the last
injection. 5-HT and 5-HIAA levels were measured using reversed phase HPLC as described
under „Materials and Methods * “ Data are plotted as a percentage of control
values in each brain region and represent the mean and S.E.M. from four to six control and
drug-treated rats. Control values for 5-HT and 5-HIAA in each of the regions were as
follows: cerebral cortex, 504 ± 58 and 422 ± 32; hippocampus, 410 ± 67 and 684 ± 89;
striaturn, 363 ± 22 and 492 ± 50; hypothalimus, 1605 ± 55 and 997 ± 42 pg/mg of
tissue, respectively.

Monoamine and metabolite
levels.
The effects of repeated systemic administration of MDMA and MDA on brain
monoamine and monamine metabolite levels were investigated at 2 weeks after the last
injection. As shown in figure 1, A and B MDMA and MDA produced marked decreases in the
content oi 5-HT and 5-HIAA in various brain regions. Both MDMA and MDA caused dramatic
decreases in 5-HIAA levels in cerebral cortex, hippocampus, striaturn and hypothalamus
(fig. 113). In hypothalamus, the reduction in 5-HIAA levels elicited by MDA was
significantly greater (P <.05) than that observed with MDMA (fig. 113). When plotted as
a percentage of control values in the respective brain regions, it was apparent that
although decreases in 5-HIAA content were observed in all brain regions examined, the
reductions in cerebral cortex and hippocampus (40-60%) were greater than those observed in
striatum and hypothalamus (30-40%). With respect to 5-HT levels, marked decreases were
observed in cerebral cortex and hypothalamus in both MDMA and MDA-treated rats (fig. 1A).
Whereas small decreases were observed in hippocampal and striatal 5-HT content after
either MDA or MDMA treatment, these reductions were found to be statistically
significant only in- -aturn (P <.Ol) of MDMA-treated rats. When calculated as “
jercentage of control 5-HT levels in the respective brain regions (fig. 1A), the data
indicate a more marked reduction in cerebral cortex (40-60%) than in hypothalamus
(18-33%).

 

In contrast with the marked and consistent effects of these compounds
on serotonergic systems, neither MDMA nor MDA administration produced any
widespread or consistent changes in levels of NE, DA, DOPAC or HVA in the various brain
regions examined; however, small changes were observed in some brain regions. Both MDMA
and MDA produced statistically significant increases in striatal DOPAC and cortical
HVA content whereas only MDMA treatment resulted in an increase in hippocampal DOPAC
levels (table 1).

 

Table 1
Effect of repeated systemic administration of MDMA and MDA on NE, DA and DA metabolite
levels in various regions of rat brain
Regional brain levels of NE, DA, DOPAC and HVA in rats 2 weeks after administration of 20
mg/kg of MDMA or MDA. Drugs were administered s.c. every 12 hr for 4 consecutive days.
Values (in picograms per milligram of tissue) represent the mean and S.E.M. of
determinations in four to six individual rats. N.D., levels were below the sensitivity of
the assay. Data were analyzed by one-way
analysis of variance and Duncans multiple range test.

Brain Region NE DA DOPAC HVA
Cerebral Cortex
Control 447 ± 53 59 ± 12 96 ± 14 19 ± 4
MDMA 424 ± 13 72 ± 3 73 ± 5 32 ± 4*
MDA 404 ± 26 63 ± 4 94 ± 19 36 ± 5*
Hippocampus
Control 528 ± 62 31 ± 9 39 ± 6 6 ± 2
MDMA 573 ± 34 13 ± 5 65 ± 12* 15 ± 5
MDA 608 ± 46 16 ± 4 32 ± 13 8 ± 5
Striatum
Control N.D. 6091 ± 596 3212 ± 159 788 ± 58
MDMA N.D. 6974 ± 228 3954 ± 320* 767 ± 46
MDA N.D. 6168 ± 569 3669 ± 189* 890 ± 48
Hypothalamus
Control 3320 ± 209 569 ± 40 228 ± 43 54 ± 4
MDMA 3052 ± 159 457 ± 38 200 ± 30 54 ± 5
MDA 3577 ± 148 585 ± 67 229 ± 26 58 ± 3

 

* Significant difference from saline-treated control group at P < .05

Monoamine uptake site density. To further determine whether changes in 5-HT and/or
5-HIAA are the consequence of long-term suppression of serotonergic function in
structurally intact neurons or whether MDMA and MDA may be affecting a
neurodegenerative process, we measured the density of monciamine uptake sites in these
brain regions to assess the structural integrity of neuronal processes. Both MDMA and MDA
caused substantial reductions in the densities of 5-HT uptake sites in all brain regions
examined. As shown in figure 2, the densities of 5-HT uptake sites are represented as a
percentage of the respective control values in cerebral cortex, hippocampus, striatum,
hypothalamus and midbrain. Significant reductions (all P < .001) were observed in
cerebral cortex (60-70%), hippocampus (70-75%), striaturn (50%), hypothalamus (40-50%) and
in midbrain (50-60%). Interestingly, MDA produced a significantly greater reduction in the
density of 5HT uptake sites in cerebral cortex than that observed with MDMA. Scatchard
analyses of [‚Hiparoxetine saturation data in control- and drug-treated rats indicated
that, in cerebral cortex, [‚H]paroxetine binding was to a single population of sites (Hill
coefficient values = 1.02, 1.01 and 1.03 in control, MDMA- and MDA-treated animals,
respectively) and that there were no significant differences in the KD values between control- and drug-treated
rats (18.8, 20.8 and 17.9 pM in control, MDMA- and MDA-treated rats, respectively).

In contrast with the effects of MDMA and MDA on the density of 5-HT
uptake sites, neither MDMA nor MDA treatment caused any significant reduction in the
levels of [‚H] mazindol-labeled NE uptake sites in cerebral cortex, hippocampus or
midbrain when compared with the respective salinetreated controls (fig. 3). Although a
small reduction was noted in NE uptake sites in hippocampus this change was not
statistically significant. Similarly, as shown in figure 4, no significant decreases were
observed in the number of [‚H] mazindol -labeled DA uptake sites in cerebral cortex,
hippocampus, striaturn and midbrain after treatment with MDA. MDMA caused a statistically
significant reduction (37%) in the density of DA uptake sites only in the midbrain. These
findings are consistent with the results from our measurements of the content of
catecholamines and catecholamine metabolites in various brain regions and support the
contention that neither MDMA nor MDA


cause any marked widespread alterations in the integrity of catecholaminergic neurons.

072.gif (25333 bytes)

Fig. 2. Effect of repeated systemic
administration of MDMA and MDA on the density of 5-HT uptake sites in various brain
regions. Rats were injected s.c. twice daily for 4 days with MDMA or MDA (20 mg/kg) or
saline vehicle (1 ml/kg) and sacrificed at 2 weeks after the last injection. Values were
determined from saturation studies in each of the regions except striaturn and
hypothalamus in which the density of 5-HT uptake sites was assessed using a saturating
concentration (0.25 nM) of [3 Hlparoxetine. No significant differences from control KI)
values (10-20 pM) were observed in either MDMA- or MDA-treated rats. Data are plotted as a
percentage of the 5-HT uptake site density observed in controls in each brain region and
represent the mean and S.E.M. from three to six rats per group. Control values were as
follows: cerebral cortex, 338 ± 10, hippocampus, 360 ± 17; striaturn, 344 ± 30;
hypothalamus, 775 ± 36; and midbrain, 570 ± 16 fmol/mg of protein. Data were analyzed by
one-way analysis of variance and Duncans multiple range test. Significant differences at P
< .001 from control values are denoted by***, whereas differences at P < .001
between MDA and MDMA treatments are denoted by t.

073.gif (45136 bytes)

Fig. 3. Effect of repeated
systemic administration of MDMA and MDA on the density of NE uptake sites in various brain
regions. Rats were injected s.c. twice daily for 4 days with MDMA or MDA (20 mglkg) or
saline vehicle (1 milkg) and sacrificed at 2 weeks after the last injection. NE uptake
sites were measured using 6 nM 13 Hlmazindol in the presence of selective blockers as
described under Waterials and Methods.“ Data are plotted as a percentage of control
values in each brain region and represent the mean and S.E.M. from six control, MDMA- and
MDAtreated animals. Control values of NE uptake sites were as follows: cerebral cortex,
164 ± 6; hippocampus, 176 ± 9; midbrain, 157 ± 13 fm61Img of protein.
Fig. 4. Effect of repeated
systemic administration of MDMA and MDA on the density of DA uptake sites in various brain
regions. Rats were injected s.c. twice daily for 4 days with MDMA or 1ADA (20 mglkg) or
saline vehicle (1 milkg) and sacrificed at 2 weeks after the last injection, DA uptake
site specific binding was determined using 6 nM [31-11mazindol in the presence of
selective blockers as described under Waterials and Methods.“ Data are plotted as a
percentage of control values in each brain region and represent the mean and S.E.M. from
six control, MDMAand MDA-treated rats. Control values were: cerebral cortex, 45 ± 7;
hippocampus, 28 ± 6; striatum, 342 ± 26; micibrain, 40 ± 3 fmollmg of protein.

 

Discussion

The present study
demonstrates that short-term administration of NIDMA or MDA for 4 days results in profound
reductions in serotonergic markers in rat brain. Decreases in 5-HT and 5HIAA content were
observed in various brain regions for up to 2 weeks after drug administration. In
contrast, MDMA and MDA produced only minor changes in catecholamine markers. These data
are consistent with other reports demonstrating that MDMA and MDA primarily alter
serotonergic function in brain (Ricaurte et al., 1985; Schmidt et al., 1986; Stone
et al., 1986), The decreases in 5-HT and 5-HIAA content after short-term
administration of MDMA and MDA may result in part from the previously reported effects of
these compounds on inhibition of tryptophan hydroxylase activity (Stone
et al., 1986) and/or their ability to increase 5-HT release (Nichols et al., 1982; Johnson et al., 1986).
However, marked decreases in 5-HT, 5-HIAA and tryptophan hydroxylase activity also have
been reported after single injections of lVIDMA or MDA (Schmidt et al., 1986; Stone et
al., 1986; Ricaurte et al., 1985). Although the changes in the serotonergic markers
described above after short-term administration of MDMA and MDA suggest neurotoxic actions
of these drugs on serotonergic neurons in brain, it is unclear whether these neurotoxic
effects represent suppressed serotonergic neurotransmission in structurally intact neurons
or whether the reductions in these markers reflect neurodegenerative processes. The
neurode generative effects of MDA on brain 5-HT terminals were postulated recently based
on decreased 5-HT levels in conjunction with histologic evidence of terminal degeneration
in striaturn (Ricaurte et al., 1985). Although the data of Ricaurte et al. (1985) provide
strong circumstantial evidence for the selective destruction of 5-HT neurons after MDA
administration, degenerati
on was assessed in only one brain region using the nonspecific Fink-Heimer
method which stains for the dege ration o various types of terminals (Fink and Heimer,
1967). Therefore, the specific loss of 5-HT terminals as well as the extent of damage to
5-HT terminals throughout the brain caused by MDMA or MDA remains to be directly
demonstrated. Inasmuch as 5-HT uptake sites are highly concentrated on 5-HT-containing
nerve terminals (Kuhar and Aghajanian, 1973), a more specific means of assessing and
quantifying a loss of 5-HT terminals would be to measure changes in the density of 5-HT
uptake sites. These sites can be selectively labeled using specific probes such as
[‚1-11paroxetine (Habert et j al., 1985). As shown in figure 2, both MDMA and MDA
cause comparable and marked reductions in the density of 5-HT uptake sites in discrete
regions of rat brain. Our contention that these changes represent the neurodegenerative
effects of these drugs on brain 5-HT terminals is supported by our preliminary
immunocytochemical data demonstrating destruction of 5-HT axons and terminals in rat brain
(O’Hearn et al., 1986). The marked reductions in 5-HT uptake sites in all the brain
regions examined in the present study would suggest that I both MDMA and MDA exhibit
little regional specificity in their ability to destroy brain 5-HT terminals. In order to
assess further the regional specificity of the neurodegenerative effects of these
compounds on serotonergic terminals, we have visualized changes in 5-HT uptake sites at
the light microscopic level after in vitro autoradiography.

Although preliminary data from autoradiographic studies confirm the drastic reductions
in 5-HT uptake sites in the brain regions used in the present study, MDMA and MDA did not
produce any marked change in the density of 5-HT uptake sites in certain brain regions
such as the lateral septum and midbrain raphe nuclei (Battaglia et al., 1987). As
there is a substantial serotonergic innervation of midbrain nuclei such as the
periaqueductal gray and substantia nigra (Steinbusch, 1983), decreases in the density of
5-HT uptake sites in midbrain seen in the present study probably represents the loss of
ser*otonergic projections to midbrain nuclei rather than the destruction of perikarya in
the raphe. Thus, there may be some neuroanatomical and regional specificity to the
neurodegenerative effects of these drugs on brain 5-HT systems.

Some subtle differences in the effects of MDMA and MDA on serotonergic parameters in
different regions of brain have been observed in the present study. For example, both MDA
and MDMA produce significant decreases in the concentration of 5-HT in cerebral cortex and
hypothalamus with little or no effect in hippocampus and striaturn. Reductions in the
density of .5-HT uptake sites in
all the brain regions examined are i Alleled more closely by similar but somewhat smaller
reductions of 5-HIAA. These data suggest that using 5-HT concentrations as an index of
degeneration of 5-HT terminals might underestimate the actual magnitude of the
neurodegenerative effect. The large disparity in correspondence between reductions in 5-HT
concentrations and the density of 5-HT uptake sites in specific brain regions (e.g., hippocampus
and striatum) that we have observed after MDMA and MDA treatment may be explained, in
part, by the increased accumulation of 5-HT that occurs in axons of passage secondary to
5-HT terminal degeneration (O’Hearn et al., 1986). Similar morphologic changes have
been reported previously in serotonergic fibers after administration of 5,6- or
5,7-dihydroxytryptamine (Baumgarten et al., 1973; Bjorklund et al., 1973).
Sensitivity differences in various brain regions to the effects of MDMA and MDA are not
unique as previous data have demonstrated differential sensitivity to the effects of
methamphetamine (Ricaurte et al., 1980) and PCA (Kohler et al., 1978; Fuller
et at., 1975) on serotonergic systems in various brain regions. Biochemical and
histochemical data suggest that PCA primarily affects the ascending 5-HT systems whereas
the descending pathways are left intact (Kohler et al., 1978; Fuller, 1978).

The neurotoxic effects of MDMA and NIDA appear to be exerted
preferentially on serotonergic neurons its no widespread changes in a variety of
catecholamine markers were seen after chronic administration of these drugs. Specifically,
no significant alteration in NE or DA concentrations were seen after administration of
MDNIA or MDA in any of the brain regions examined; the effects of MDA and MDMA
administration on the concentrations of DOPAC and HVA were more variable. Small but
significant increases in DOPAC and/or HVA were seen in cerebral cortex, hippocampus and
striaturn after chronic administration of these methylenedioxyamphet.ainine derivatives.
Similar increases in DA metabolite levels, reflecting increases in DA turnover, have been
observed in discrete regions of brain after both acute (Schmidt et al., 1986) and
chronic (Stone et al., 1986) administration of MDMA and MDA. Because
5-HT-containing terminals are present in high concentrations in midbrain areas (substantia
nigra and ventral tegmental area) containing DA cell bodies (Steinbusch, 1983), tile
degeneration of 5-HT terminals in these regions after MDMA or MDA treatment may be
responsible for the observed changes in DA metabolites. Despite these small effects on DA
turnover, MDMA and MDA do not appear to produce any widespread destruction of
catecholaminergic terminals as the only change observed was a small reduction in DA uptake
sites in midbrain after administration of MDMA. The reasons for the decrease in DA uptake
sites are at present unclear. Preliminary immunocytochemical data indicate that there are
no changes in the density or morphology of catecholarnine axons after chronic
administration of MDMA or MDA (O’Hearn et al., 1986). We are presently carrying out
detailed autoradiographic studies to identify any discretely localized small change in
catecholaminergic neurons.

 

In summary, the data we present here demonstrate that repeated, systemic administration
of either MDMA or MDA can cause profound and preferential destruction of 5-HT terminals in
various regions of rat brain. The use of in vitro radioligand binding techniques to
measure the density of monoamine uptake sites provides a means for quantification of the
extent of degeneration of 5-HT terminals. This approach in conjunction with
„conventional“ measures of reductions in monoamine and monoamine metabolite
levels allow us to conclude that whereas MDA and MDMA are without any widespread long-term
effects on the functional and structural integrity of catecholamine neurons, these
compounds cause a longlasting degeneration of 5-HT neurons in a number of regions of rat
brain. The physiological consequences and compensatory mechanisms in response to the
long-term destruction of serotonergic neurons in brain remain to be assessed.

 

ABBREVIATIONS: MDMA,
3,4-methylenedioxymethamphetamine; MDA,
3,4-methylenedioxyamphetamine; DA, dopamine; 5-HT, serotonin; 5-HIAA,
5-hydroxyindoleacetic acid; NE, norepinephrine; HPLC, high-performance liquid
chromatography; DHBA, dihydroxybenzyiamine; DOPAC, 3,4
dihydroxy phenyl acetic acid; WA, homovanillic acid; DMI, desipramly~l~“.
-chioroamphetamine.


Acknowledgments
The authors thank Brian Kuyatt for excellent technical assistance, Drs. Charles Haertzen
and Lawrence Thompson for help with data analysis andTerrie Pierce and Mary Flutka for
manuscript preparation.

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