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Behavioural Brain Research 189 (2008) 191201

Research report

Sub-chronic nandrolone treatment modifies neurochemical

and behavioral effects of amphetamine and
3,4-methylenedioxymethamphetamine (MDMA) in rats
Sanna Kurling , Aino Kankaanpaa , Timo Seppala
Drug Research Unit, Department of Mental Health and Alcohol Research,
National Public Health Institute, Helsinki, Finland
Received 26 June 2007; accepted 28 December 2007
Available online 5 January 2008

Abstract
Misuse of anabolic-androgenic steroids (AASs) is increasing, and appears to have much in common with the use of substances known
to induce drug dependence. Moreover, persons who abuse AASs also tend to abuse other psychotropic drugs such as amphetamine or 3,4-
methylenedioxymethamphetamine (MDMA, ecstasy). The aim of this study was to investigate whether nandrolone (5 5 or 5 20 mg/kg)
pre-exposure modulates the acute neurochemical and behavioral effects of amphetamine (1 mg/kg) and MDMA (5 mg/kg) in rats. Dopamine (DA),
5-hydroxytryptamine (5-HT) and their metabolites were measured from samples collected from the nucleus accumbens (NAc) by in vivo microdial-
ysis. The behavior of the animals was recorded on videotapes, from which it was later rated. Our results demonstrate that sub-chronic treatments
with supraphysiological doses of nandrolone attenuate dose-dependently the increase in extracellular DA concentration evoked by amphetamine
or MDMA. The lower dose of nandrolone attenuated MDMA-induced increase in 5-HT-levels, while the higher dose potentiated it. Analysis of the
behavioral data suggests that effects of the amphetamine and MDMA are dose-dependently attenuated by AAS-treatment, paralleling DA results.
In conclusion, the results of this study show that AAS-pre-treatment is able to modulate the reward-related neurochemical and behavioral effects
of amphetamine and MDMA.
2008 Elsevier B.V. All rights reserved.

Keywords: Amphetamine; Anabolic steroids; Dopamine; MDMA; Nucleus accumbens; 5-HT

1. Introduction humans: Changes in mood, euphoria, delusions, depression and

A large number of adolescents abuse anabolic-androgenic
steroids (AASs) to improve their physical fitness and appear-
ance. However, this misuse of AASs probably involves more
than a desire to enhance the users appearance or sporting per-
formance: it appears to have much in common with the use of
alcohol and tobacco [30]. Indeed, animal studies have indicated
that AASs are able to evoke biochemical changes in dopaminer-
gic and serotonergic neuronal systems related to reward, a key
phenomenon in drug dependence, as well as numerous other
behavioral responses in rats [7,50,51]. This is in line with the
behavioral effects associated with prolonged use of AASs in
Corresponding author at: Drug Research Unit, National Public Health Insti-
tute, Mannerheimintie 166, FIN-00300, Helsinki, Finland.
Tel.: +358 9 4744 8856; fax: +358 9 4744 8553.
E-mail address: sanna.kurling@ktl.fi (S. Kurling).
violent behavior [3]. In addition, it has been suggested that mis-
use of AASs may serve as a gateway to the abuse of other
substances [2].
According to recent surveys, people who abuse AASs also
tend to abuse other psychotropic drugs such as cocaine, heroin,
amphetamine and 3,4-methylenedioxymethamphetamine
(MDMA, ecstasy) [18,29], a phenomenon that may con-
tribute to aggressive behavior. Amphetamine and MDMA are
drugs most popularly used with anabolic steroids after cannabis
in Sweden [40]. One explanation for the popularity of stimulant
drugs among steroid users is that they induce sensation of an
energy burst that enables one to train even harder.
The aim of this study was to evaluate whether AAS-pre-
treatment induces changes, measurable as altered response to
drugs of abuse, in brain reward circuitry. We used in vivo micro-
dialysis in fully conscious rats to monitor whether sub-chronic
nandrolone (5 5 or 5 20 mg/kg i.m.) treatment modulates

0166-4328/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2007.12.021
192 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201
the effects of acute injections of amphetamine (1 mg/kg i.p.)
or MDMA (5 mg/kg i.p.) on extracellular levels of dopamine
(DA) and 5-hydroxytryptamine (5-HT) and their metabolites
3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid
(HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus
accumbens (NAc), which is implicated as a major locus in brain
dopaminergic reward circuitry. During the microdialysis exper-
iments the animals behavior was recorded on videotapes, from
which the behavior was later assessed by an observer blind to
drug conditions. In addition, brain and/or plasma concentrations
of amphetamine and MDMA were measured to evaluate whether
the neurochemical and behavioral effects observed were due to
nandrolone-induced changes in stimulant pharmacokinetics.
2. Materials and methods
2.1. Animals
Adult male Wistar rats, weighing 300380 g, were used in this study. The
animals were supplied by Harlan Netherlands B.V. (the Netherlands) at least 1
week before the experiments, and they were housed in groups of three in Tech-
niplast Eurostandard type IV cage (595 mm 380 mm 200 mm, floor area
1820 cm2 ) in a temperature (21 2 C) and humidity controlled room with a
12-h light cycle. The lights were on from 06.00 a.m. to 06.00 p.m., during which
time all the experiments were conducted. Standard laboratory chow (Altromin
Nr. 1314; Chr. Petersen A/S, Ringsted, Denmark) and tap water were freely
available. After surgery, the rats were housed individually. The local institu-
tional committee for animal care and use and the chief veterinary surgeon of the
county administrative board approved the animal experiments, and they were
conducted according to the European Convention for the Protection of Vertebrate
Animals used for Experimental and other Scientific Purposes.
2.2. Drugs and treatments
Nandrolone decanoate (Deca-Durabolin ) was a commercial preparation
from NV Organon (Oss, the Netherlands). The matching vehicle for Deca-
Durabolin , a mixture of arachinoid oil and benzylalcohol, was prepared by
the University Pharmacy (Helsinki, Finland) for control purposes. Nandrolone
was administered in doses of 0 (vehicle), 5, and 20 mg/kg (calculated as free
base) intramuscularly (i.m.) every other day during a 10-day period (total of 5
injections per animal). The injections were given in the left and right hind leg
alternately.
3,4-Methylenedioxymethamphetamine HCl (National Institute on Drug
Abuse; NIDA, Bethesda, MD, USA) and amphetamine sulfate (Sigma Chemical
Co., St Louis, MO, USA) were dissolved in saline (0.9% NaCl) at concen-
trations of 5 and 1 mg/kg (calculated as free base). Both drugs were racemic
mixtures. The drugs were injected intraperitoneally (i.p.) in a volume of 1 ml/kg
of body weight during the microdialysis experiment, 6 days from last nandrolone
injection. The animals were weighed before each injection to adjust the exact
dose.
The doses of nandrolone, as well as amphetamine and MDMA, were chosen
to correspond to doses taken by human abusers, as calculated per weight. The two
nandrolone doses, 20 and 5 mg/kg, were chose to parallel human heavy users
doses; the typical AAS abusers are reported to use nandrolone decanoate
5.418.6 mg/kg (i.m.) daily during 2-week period, in a cocktail with other
anabolic steroids [21], whereas the clinical dose is markedly lower, namely
50200 mg daily during a 2-week period (0.72.8 mg/kg; 70 kg human adult
patient). The normal clinical oral dose of amphetamine is 0.070.2 mg/kg [4],
while addicts may self-administer doses as high as 0.74.3 mg/kg. Typical
amount of MDMA abused by humans is one or two tablets of 80100 mg
orally, which corresponds to 13 mg/kg [23]. In addition to comparability to
human doses, also experiences from our earlier animal studies were taken into
account: a 1-mg/kg dose of amphetamine and a 5-mg/kg dose of MDMA both
produce robust and reproducible neurochemical and behavioral effects without
any observable adverse effects on animal welfare.
2.3. Microdialysis surgery and experiments
The rats were anesthetized with 5% halothane gas (Halothane Liquid
BP; Rhodia Organique Fine Ltd., Bristol, UK) and placed in a stereotactic
instrument. During surgery halothane gas was administered at a concentra-
tion of 2.5%. A burr hole was drilled for a guide cannula (CMA/12; CMA
Microdialysis, Solna, Sweden), which was implanted 2 mm above the NAc
[A, +1.9; L, 1.0; D, 6.0 as calculated relative to the bregma and skull
surface according to Paxinos and Watson [42]]. The cannula was secured
with two small screws and dental cement (Aqualox; VOCO, Cuxhaven,
Germany). The animals received s.c. 0.05 ml of buprenorphium prepara-
tion (Temgesic , 0.3 mg/ml; Schering-Plough Europe, Brussels, Belgium)
to alleviate the pain, and were allowed to recover from the surgery for 5
days.
One day before the experiment, the rats were allowed to habituate to test
cages and a microdialysis probe (CMA/12, membrane length 2 mm; CMA
Microdialysis) was inserted through the guide cannula into the NAc. On the
day of the experiment, the animals were placed in test cages and probes
were connected to a CMA/100 microinjection pump and perfused with mod-
ified Ringers solution (147 mM NaCl, 1.2 mM CaCl2 , 2.7 mM KCl, 1.0 mM
MgCl2 , pH 6) at a flow rate of 2 l/min. In order to prevent degradation of
monoamine transmitters, a 6.5-l aliquot of an aquaeous antioxidant solution
(1.0 mM oxalic acid, 3.0 mM l-cysteine, 0.1 mM Acetic acid) was added to each
vial before collecting the dialysate samples [27]. The perfusate from the first
60 min was discarded, after which the samples were collected at 20 min inter-
vals. Amphetamine, MDMA or saline were injected i.p. after collection of four
basal samples (2 h 20 min from beginning of the perfusion). Animal behavior was
video recorded from 20 min before injection up to 220 min after the injection. At
the end of the experiment the animals were anesthetized with 5% halothane gas,
and blood samples for drug measurements were drawn using cardiac puncture.
After decapitation, their brains were dissected out and immersed in buffered
10% formalin solution to verify the correct placement of the probes. Only
data from animals with accurate probe placements were included in statistical
analyses.
The interval between last nandrolone injection and the microdialysis exper-
iment was chosen to ensure proper recovery period after the surgery. To
evaluate whether the blood nandrolone concentration remained on adequate
levels, we performed a preliminary experiment, in which nandrolone concen-
trations were measured from tail vein blood samples drawn on days 1, 2, 4,
8 and 16 after a single 20-mg/kg injection of nandrolone decanoate (n = 6).
Nandrolone concentrations measured were as follows: day 1 = 1.69 0.29 g/l,
day 2 = 1.59 0.28 g/l, day 4 = 2.58 0.33 g/l, day 8 = 1.67 0.29 g/l, day
16 = 1.26 0.21 g/l).
2.4. Analytical procedures
In order to quantitate DA, 5-HT and their metabolites DOPAC, HVA and
5-HIAA, 20-l aliquots of dialysate samples were injected into an ESA (ESA
Inc. Chelmsford, MA, USA) high performance liquid chromatography (HPLC)
apparatus equipped with an Inertsil ODS-3V 5 m (4.6 mm 250 mm i.d.)
reverse-phase column (GL-Sciences Inc., Tokyo, Japan) and a coulometric ESA
Coulochem III detector. The mobile phase was a mixture of a buffer contain-
ing 50 mM NaH2 PO4 , 0.1 mM Na2 EDTA, 2.3 mM octanesulfonic acid, and
acetonitrile (14%, v/v in the final solution), with the pH adjusted to 3.0 with
orthophosphoric acid (H3 PO4 ). The mobile phase was filtered through a 47 mm
Hydrophilic Polypropylene Membrane filter with pore size of 0.22 m (Gelman
Sciences, Ann Arbor, MI, USA) and degassed under vacuum. The flow rate was
1.2 ml/min and the detector potentials of the two electrodes were 175 mV and
+250 mV.
Amphetamine and MDMA concentrations were analyzed with gas
chromatographymass spectrometry (GCMS) by using a method described
earlier [26]. Briefly, amphetamine and MDMA were extracted from blood,
and derivatized with heptafluorobutyric anhydride (HFBA) as follows:
200 l of blood was mixed with 50 l of alkaline buffer and 500 l of
extractionderivatization reagent (toluene + HFBA + internal standard), cen-
trifuged, and injected into a GCMS apparatus. When analyzing brain tissue
samples, the procedure was begun as follows: The deep frozen rat brains were
 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201
dissected sagittally along the fissura longitudinalis cerebri, the left hemispheres
were weighed and homogenized in 4 ml of 0.1 M HClO4 . After centrifugation,
200-l aliquots of the supernatants were transferred to clean test tubes and the
extractionderivatization was performed according to the procedure described
for blood samples.
Blood nandrolone concentrations were measured with GCMS as deriva-
tives formed with N-methyl-n-(trimethylsilyl)trifluoroacetamide (MSTFA;
SigmaAldrich Co. Ltd., Dorset, UK). Working standards were prepared
from nandrolone (19-nortestosterone) purchased from Fluka Chemie GmbH
(Buchs, Switzerland). The internal standard (IS), isotope labeled tetrahy-
drocannabinol (THC-D3) was from Cerilliant (Round Rock, TX, USA).
Nandrolone was extracted from blood samples as follows: 5 ml of toluene
containing IS was vortexed with 0.5 ml of the sample. After centrifugation,
the toluene layer was transferred into a clean test tube and evaporated to
dryness. The dry residue was dissolved in 60 l of butyl acetate, and nan-
drolone (and IS) was derivatized with 15 l of MSTFA. A 2-l aliquot of
the mixture was injected into an Agilent GCMS (EI) apparatus equipped
with a DB-35MS capillary column of length 30 m, internal diameter 320 m
and film thickness 0.25 m (J&W Scientific Inc., Folsom, CA, USA). The
oven temperature was initially 150 C with a hold time of 1.0 min, and
was increased 15 C/min to 320 C, with a final hold time of 3.0 min. The
lower limit of quantitation was set at 0.5 g/l. At a concentration level of
1.0 g/l, bias (%) and precision (R.S.D.%) were 1.0 and 3.5%, respec-
tively.
2.5. Characterization of behavioral changes
The behaviors of the rats during the microdialysis experiments were char-
acterized from videotapes by an observer blind to drug conditions. Recording
was begun 20 min before injection and continued for 160 min after the injec-
tion. Monitoring was discontinued for 20 min following the injection to exclude
the effect of the injection. Locomotor activity (LMA) of the animals was esti-
mated from the number of complete passes across midline of the test cage at
intervals of 20 min (corresponding to the sampling interval in the microdialysis
experiments).
In addition, a more detailed behavioral analysis was performed at the same
time intervals. The beginning, frequency, and/or duration of different behav-
ior were monitored visually for 1 min every 5th min. The behavioral patterns
were scored according to a rating scale modified from the one used in our
previous study [25] and the one described by Chin et al. [11]. The rats were
given a single behavioral score per each 1 min observation point and mean
values were calculated for each 20-min sampling interval. Behavioral scores
were: 0, passive motionlessness; 1, active motionlessness; 2, active motion-
lessness with occasional movements; 3, sniffing, grooming, occasional LMA;
4, LMA with burst of rearings, slight agitation; 5, stereotyped behavior; 6,
intense stereotyped behavior; 7, ataxia. When rated as passive motionless the ani-
mal was stationary, lying or sleeping, whereas active motionlessness included
alertness. Active motionlessness with occasional movements was defined as
movements of the head or occasional movement of the animal. LMA was
defined as movement of the animal over the surface of the observation cage
floor. Stereotyped behavior was defined as compulsive-like, rapid, and repet-
itive purposeless behavior, such as intensive sniffing (e.g. during a prolonged
rearing), head or body weaving or head bobbing. Ataxia was defined accord-
ing to Sturgeon et al. [49] as impairment in the ability of the animal to
execute coordinated motor responses leading, in the extreme, to incapacita-
tion.
2.6. Statistics
In the microdialysis experiments the mean of the four samples before the
drug treatments was considered to represent basal release (100%) of neuro-
transmitters and their metabolites according to which relative changes after
the injections were calculated. The absolute basal releases were calculated on
the real values. In the LMA test the absolute number of passes across mid-
line was counted, and for the more detailed behavioral analysis, scoring was
conducted as described above. For statistical evaluations, both neurochemical
and behavioral data were calculated as areas under the curves (AUCs) with
193
the trapezoidal method. The microdialysis data were then subjected to a 2-
tailed t-test, or one-way analysis of variance (ANOVA) followed by Tukeys
test. The behavioral scores were analyzed with the MannWhitney U test, or
KruskallWallis nonparametric ANOVA followed by the MannWhitney U test
with Tukeys adjustment for multiple comparisons. The results from measure-
ments of MDMA blood concentrations were analyzed with one-way ANOVA
followed by Tukeys test. The results are presented as means S.E.M. (stan-
dard error of the mean) and the level of statistical significance was set at
p 0.05.
3. Results
3.1. Effects of nandrolone pre-treatment on amphetamine-
or MDMA-induced neurochemical changes
The absolute basal values of the different treatment groups
did not differ significantly. The means (S.E.M.; n = 54) of
the basal concentrations in the NAc dialysate were as follows:
DA 12.6 1.1 fmol/40 l; 5-HT 7.4 0.9 fmol/40 l; DOPAC
18.0 1.3 pmol/40 l; HVA 7.5 0.5 pmol/40 l and 5-HIAA
8.8 0.3 pmol/40 l. The means of the first and the last (1
and 4) basal sample concentrations did not differ more than
6.8%. Sub-chronic i.m. pre-treatment with nandrolone at doses
of 5 and 20 mg/kg did not affect spontaneous release of the
transmitters (Fig. 1) or their metabolites. The basal levels
also remained unaltered after acute saline injection during the
microdialysis experiments (Fig. 1). I.p administration of either
amphetamine (1 mg/kg) or MDMA (5 mg/kg) elevated extra-
cellular DA levels when compared with saline-treated animals,
as revealed by the t-test: p < 0.001, amphetamine; p < 0.001,
MDMA). Amphetamine- or MDMA-treatment also elevated
the extracellular 5-HT-levels with statistical significance com-
pared with saline-treated animals (p = 0.037, amphetamine;
p = 0.001, MDMA; t-test). In addition, administration of either
amphetamine or MDMA decreased DOPAC levels (p = 0.001,
amphetamine; p = 0.001, MDMA; t-test) as well as HVA
concentrations (p = 0.027, amphetamine; p = 0.021, MDMA;
t-test).
As evident from Fig. 2, pre-treatment with nandrolone
decreased with statistical significance the amphetamine-induced
elevation of extracellular DA levels (p = 0.001, AUC; ANOVA)
in the NAc. Fig. 2 also summarizes the effects of nandrolone
pre-treatment on amphetamine-induced decrease in DOPAC
levels. The one-way ANOVA revealed a significant nandrolone-
amphetamine interaction (p = 0.002). As seen in Figs. 2 and 3,
the pre-treatment did not have a significant effect on extracellular
levels of HVA, 5-HT and 5-HIAA after amphetamine injection
compared with vehicle pre-treated animals.
Nandrolone pre-treatment significantly decreased the
MDMA-induced elevation of extracellular DA compared
with vehicle treated animals (p = 0.001; ANOVA; Fig. 4).
Nandrolone treatment also modified the effects of MDMA on
extracellular DOPAC (p = 0.001, ANOVA) and HVA (p < 0.047,
ANOVA) levels (Fig. 4). The effects of both nandrolone doses
on MDMA evoked changes in extracellular levels of 5-HT and
5-HIAA that are presented in Fig. 5. Nandrolone pre-treatment
altered the effects of MDMA on extracellular 5-HT (p = 0.001;
ANOVA), but not 5-HIAA-levels. The lower dose of nandrolone
194 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201

Fig. 1. The effects of amphetamine (1 mg/kg) MDMA (5 mg/kg) and saline on extracellular DA and 5-HT and their metabolite levels in the NAc. The times of
the injections are indicated by arrows. Data expressed as percentages of basal release are given as means S.E.M. (n = 6). Histograms represent the area under the
curve (AUC) after injection of the drug, and the minutes where AUC is calculated are apparent in the figure. # p 0.05, ## p 0.01, ### p 0.001 compared with
vehicle-saline group, Tukeys test. V/S = vehicle + saline, N5/S = nandrolone 5 mg/kg + saline, N20/S = nandrolone 20 mg/kg + saline, V/A vehicle + amphetamine,
V/MDMA = vehicle + MDMA.

attenuated MDMA-induced increase in 5-HT-levels, while the
higher dose potentiated it. Both these changes were statistically
significant (see Fig. 5).
3.2. Effects of nandrolone pre-treatment on amphetamine-
or MDMA-induced behavioral changes
Nandrolone pre-treatment per se or acute saline injections
did not alter the behavior of the rats, while administration of
amphetamine or MDMA had profound effects on both LMA
and stereotyped behavior. As shown in Fig. 6, administration
of amphetamine or MDMA increased significantly both the
behavioral scores (p = 0.004, amphetamine; p = 0.004, MDMA;
MannWhitney U test) and LMA (p = 0.017, amphetamine;
p = 0.023, MDMA; t-test), compared with saline. Amphetamine-
induced behavioral patterns are clearly distinguishable from
those of the saline-treated rats. While saline-treated ani-
mals were mostly sleeping or awake but motionless, the
amphetamine-treated were exhibiting behavioral patterns such
as increased moving with burst of rearings, slight agitation,
stereotyped behavior such as intensive sniffing, head or body
weaving or head bobbing. Behaviors of the MDMA-treated
animals resembled mostly those of the amphetamine-treated ani-
mals except that MDMA treated animals exhibited more LMA.
Nandrolone pre-exposure modified the ability of
amphetamine to increase LMA as seen in Fig. 7 (p < 0.001;
ANOVA). The effects of nandrolone pre-treatment on behav-
ioral patterns induced by amphetamine paralleled those seen
in LMA results, (p < 0.001 AUC; KruskallWallis). Less
stereotyped behavior was observed; the frequency and duration
of rapid and repetitive purposeless behavior and intensive
sniffing were reduced, head and body weaving were not so
broad, and head bobbing was no longer observed.
Nandrolone pre-exposure also modified the ability of MDMA
to increase LMA as seen in Fig. 7 (p = 0.008; ANOVA). Fol-
lowing MDMA injection, nandrolone pre-treated rats had lower
behavior scores than those treated with vehicle (p = 0.003 AUC;
p = 0.004 PEAK; KruskallWallis). Overall, the alterations in
behavior were very similar to those observed in animals treated
with nandrolone and amphetamine.
 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201 195

Fig. 2. The effects of sub-chronic nandrolone and acute amphetamine (1 mg/kg) injections on extracellular DA, DOPAC and HVA levels in the NAc. The times
of the amphetamine injections are indicated by arrows. Data expressed as percentages of basal release are given as means S.E.M. (n = 6). Histograms represent
the area under the curve (AUC) after injection of the drug, and the minutes where AUC are apparent in the figure. * p 0.05, ** p 0.01, *** p 0.001, Tukeys
test. Vehicle-saline group is added in picture as baseline reference. V/A = vehicle + amphetamine, N5/A = nandrolone 5 mg/kg + amphetamine, N20/A = nandrolone
20 mg/kg + amphetamine.

Fig. 3. The effects of sub-chronic nandrolone and acute amphetamine (1 mg/kg) injections on extracellular 5-HT and 5-HIAA levels in the NAc. The times of
the amphetamine injections are indicated by arrows. Data expressed as percentages of basal release are given as means S.E.M. (n = 6). Histograms represent
the area under the curve (AUC) after injection of the drug, and the minutes where AUC are apparent in the figure. *p 0.05, **p 0.01, ***p 0.001, Tukeys
test. Vehicle-saline group is added in picture as baseline reference. V/A = vehicle + amphetamine, N5/A = nandrolone 5 mg/kg + amphetamine, N20/A = nandrolone
20 mg/kg + amphetamine.
196 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201

Fig. 4. The effects of sub-chronic nandrolone and acute MDMA (5 mg/kg) injections on extracellular DA, DOPAC and HVA levels in the NAc. The times of the
amphetamine injections are indicated by arrows. Data expressed as percentages of basal release are given as means S.E.M. (n = 6). Histograms represent the area
under the curve (AUC) after injection of the drug, and the minutes where AUC are apparent in the figure. * p 0.05, ** p 0.01, *** p 0.001, Tukeys test. Vehicle-
saline group is added to the figure as a baseline reference. V/MDMA = vehicle + MDMA, N5/MDMA = nandrolone 5 mg/kg + MDMA, N20/MDMA = nandrolone
20 mg/kg + MDMA.

Fig. 5. The effects of sub-chronic nandrolone and acute MDMA (5 mg/kg) injections on extracellular 5-HT and 5-HIAA levels in the NAc. The times of the
amphetamine injections are indicated by arrows. Data expressed as percentages of basal release are given as means S.E.M. (n = 6). Histograms represent the
area under the curve (AUC) after injection of the drug, and the minutes where AUC are apparent in the figure. * p 0.05, ** p 0.01, *** p 0.001, Tukeys test.
Vehicle-saline group is added in picture as baseline reference. V/MDMA = vehicle + MDMA, N5/MDMA = nandrolone 5 mg/kg + MDMA, N20/MDMA = nandrolone
20 mg/kg + MDMA.
 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201 197

Fig. 6. The effects of the amphetamine and MDMA on locomotor activity and behavior. The rats were given a single behavioral score per each 1 min observation
point and mean values were calculated per each 20-min sampling interval. Behavioral scores were: 0, passive motionlessness; 1, active motionlessness; 2, active
motionlessness with occasional movements; 3, sniffing, grooming, occasional locomotor activity; 4, locomotor activity with burst of rearings, slender agitation; 5,
stereotyped behavior; 6, intense stereotyped behavior; 7, ataxia. Activity of the animals was estimated from the number of complete passes across the midline of the
test cage during periods of 20 min (corresponding to the sampling interval in the microdialysis experiments). Amphetamine (1 mg/kg), MDMA (5 mg/kg) or saline
was administered at 0 min and is indicated by arrows. Data expressed as absolute behavioral scores and passes across midline (n = 6). Histograms represent AUC 0
to 140 min effect after injection of the drug. # p 0.05, ## p 0.01, ### p 0.001 compared with vehicle-saline group, t-test. V/S = vehicle + saline, N5/S = nandrolone
5 mg/kg + saline, N20/S = nandrolone 20 mg/kg + saline, V/A = vehicle + amphetamine, V/MDMA = vehicle + MDMA.

3.3. Effects of nandrolone pre-treatment on amphetamine
or MDMA concentrations
According to our preliminary study of one brain sample per
treatment group, sub-chronic i.m. pre-treatment with nandrolone
at doses of 5 and 20 mg/kg caused only negligible changes in
brain tissue amphetamine concentration: results for all treatment
groups were between 3.0 and 4.0 g/g, with the lowest concen-
tration measured for the group that received no nandrolone. In
case of MDMA, however, the preliminary study showed more
variable results: the MDMA brain concentrations in animals
treated with arachinoid oil, nandrolone 5 mg/kg, and nandrolone
20 mg/kg were 5.2, 1.7, and 2.0 g/g, respectively. Therefore,
MDMA was also measured from blood samples from 19 animals
that were included in the neurochemical and behavioral study.
The means of the MDMA blood concentrations in all treatment
groups were between 0.10 to 0.11 g/ml, which suggests that
nandrolone treatment does not change MDMA pharmacokinet-
ics.
4. Discussion
One of the main findings of the present study is
that sub-chronic pre-treatment with supraphysiological doses
of nandrolone attenuate dose-dependently amphetamine- or
MDMA-induced increase in extracellular DA in the NAc. As
expected, a similar trend of attenuation of the stimulant effects
was also apparent in the extracellular levels of DA metabolites
DOPAC and HVA. Given that activation of the dopaminer-
gic neuronal system in mesolimbic brain areas, including the
NAc, is thought to play a major role in mediating the reward-
ing properties of drugs of abuse [52], our findings are in line
with the reported ability of nandrolone to decrease morphine-
reward [10] as well as preliminary findings [37] indicating that
chronic administration of AASs blunts the subjective effects of
cocaine in rats. Chronic nandrolone treatment can alter some of
the effects of drugs of abuse, at least cannabinoids [9]. Combi-
nations of nandrolone and testosterone derivatives has also been
shown to decrease c-fos response to morphine in rat brain [24],
but also contrary results has been published [13]. Taken together,
these results suggest that AASs may induce adaptations in brain
reward systems.
According to our results, nandrolone has dose-related atten-
uating effect on amphetamine-induced elevation of extracellular
DA levels in the NAc, while in the study of Birgner et al. [6]
there was no statistically significant change in an apparently
similar experimental set-up. Although the explanation for dis-
crepant results is not obvious, a closer examination reveals that
there are differences both in treatment regimens and methodol-
ogy in these two studies. For example, we have examined the
neurochemistry and behaviors 6 days after the last treatment
with nandrolone, while Birgner and coworkers have used an
interval of only 24 h. Birgner et al. have also treated their rats
with 14 15 mg/kg of nandrolone, while our rats in the high
dose group received 5 20 mg/kg. Given that accumulation of
nandrolone following administration of an oil-based preparation
is considerable, the treatment regimen of Birgner and coworkers
must have yielded markedly higher nandrolone plasma concen-
trations than ours. They have also used five times higher dose
of amphetamine than we did. Therefore, the two studies are
not completely comparable. It must also be noted that although
Birgner and coworkers did not observe a statistically significant
alteration in amphetamine induced elevation of extracellular DA
levels after nandrolone treatment, a clear trend toward decrease
can be seen in their results.
Concerning the mechanisms by which AASs may modu-
late the brain reward systems, it can be speculated whether
their actions might be targeted to DA synthesis. The decreased
amount of DA available would explain the diminished response
for amphetamine or MDMA, which are both considered to exert
198 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201

Fig. 7. The effects of the amphetamine and MDMA on locomotor activity and behavior. The rats were given a single behavioral score per each 1 min observation
point and mean values were calculated per each 20-min sampling interval. The behavioral scores were: 0, passive motionlessness; 1, active motionlessness; 2, active
motionlessness with occasional movements; 3, sniffing, grooming, occasional locomotor activity; 4, locomotor activity with burst of rearings, slender agitation;
5, stereotyped behavior; 6, intense stereotyped behavior; 7, ataxia. Activity of the animals was estimated from the number of complete passes across midline of
the test cage during periods of 20 min (corresponding to the sampling interval in the microdialysis experiments). Amphetamine (1 mg/kg) or MDMA (5 mg/kg)
was administered at 0 min and is indicated by arrows. Data are expressed as absolute behavioral scores and passes across midline (n = 6). Histograms represent
the area under the curve (AUC) and the minutes where AUC is calculated are apparent in the figure. * p 0.05, ** p 0.01, *** p 0.001, MannWhitney U test
in LMA and t-test in behavior scores. V/A = vehicle + amphetamine, N5/A = nandrolone 5 mg/kg + amphetamine, N20/A = nandrolone 20 mg/kg + amphetamine,
V/MDMA = vehicle + MDMA, N5/MDMA = nandrolone 5 mg/kg + MDMA, N20/MDMA = nandrolone 20 mg/kg + MDMA.

their rewarding effects by increasing the extracellular levels of
DA in NAc specially in the shell [8]. However, although there are
findings, such as decrease in TH mRNA levels [22] or increase
in expression of DA autoreceptor mRNA [31], which may sug-
gest a decrease in DA synthesis following AAS-treatment, such
explanation seems unlikely, because we found no change in
the absolute basal levels of extracellular DA following AASs
pre-treatment. Even if the unaltered basal DA-levels seen here
resulted from compensatory mechanisms, our previous study
showed that the accumbal tissue content of DA remained unal-
tered in AAS treated animals [34]. Thus, it seems that this step of
DA transmission is not the primary target of nandrolone action.
Amphetamine and MDMA are well known to increase
extracellular dopamine levels by reverse transport via DA trans-
porter (DAT). Therefore, changes in either amount of DAT,
or its functioning, would result in changes in DA response
for amphetamine-like compounds. Indeed, chronic nandrolone
treatment has been reported to increase both the amount of DAT
and its affinity to DA [28,32]. However, in addition to their abil-
ity to release DA, both amphetamine and MDMA are also able
to inhibit DA reuptake mechanism mediated by DAT, which
markedly complicates assessment of any actions on DAT by
nandrolone.
Inside the cells, transportation of DA into or out to the stor-
age vesicles is mediated by the CNS vesicular monoamine
transporter (VMAT-2), which modulates the concentration of
releasable DA in the nerve terminals [20]. Thus, modification
in the action of VMAT-2 by AASs would change DA response
to amphetamine. There is evidence that chronic treatment with
ovarian steroids can lead to a decrease in VMAT-2 mRNA lev-
els in the NAc [43]. Taken that we did not observe any changes
in accumbal DA tissue content in our previous study [34], the
decrease in the amount of releasable DA might be a plausible
explanation for the attenuated effect of amphetamine or MDMA.
At the dose used, amphetamine, given with or without
nandrolone pre-treatment, had only a negligible effect on
 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201
extracellular 5-HT or 5-HIAA levels. Instead, the effects of
nandrolone on MDMA-induced elevation of 5-HT levels were
two sided: 5-mg/kg dose of nandrolone decreased the effect of
MDMA, while the 20-mg/kg dose potentiated it. MDMA causes
an acute and rapid increase in extracellular 5-HT through release
from synaptic vesicles, blockade of 5-HT reuptake via presynap-
tic serotonin transporter (SERT) and inhibition of monoamine
oxidase [39]. MDMA releases 5-HT from striatal slices at con-
centrations that are 10-fold lower than concentrations required
for stimulating DA release [45,47]. Our results are in line with
that. Again, a massive decrease in 5-HT biosynthesis can be
excluded, because the absolute basal levels were equal in AAS
pre-treated animals and vehicle pre-treated animals, as were the
tissue concentrations in our previous study [34]. In addition,
testosterone treatment had been shown to lack any effect on
tryptophan hydroxylase activity in limbic system [33].
Damage to 5-HT terminals following high doses of MDMA
is well documented [41,44]. MDMA neurotoxicity is preceded
by an acute stage in which substantial release of 5-HT leads to
exhaustion of the neuronal 5-HT stores. An apparently similar
outflow of 5-HT was observed after MDMA injection in our
rats pre-treated with the higher dose of nandrolone. Therefore,
it can be speculated whether high-dose sub-chronic nandrolone
treatment might increase cell vulnerability to MDMA, leading
to effects resembling acute toxicity at lower concentrations of
MDMA. Concerning the mechanisms by which these changes
might occur, the free radical formation in the brain has been con-
sidered a key phenomenon in acute toxic effects of MDMA in
experimental animals. Sex steroids may play a role in free radi-
cal formation, for example, in mammals, sex steroids control the
expression of the neuronal nitric oxide (nNOS) in the pre-optic-
hypothalamic region. Because castration is shown to decrease
the number of neuronal nitric oxide synthase-immunoreactive
(nNOS-IR) neurons in male rats [17], it is tempting to spec-
ulate whether repeated administration of high dose anabolic
steroid might have a reverse effect. In addition, high concen-
trations of testosterone are able to initiate neuronal cell death
[19]. However, it must be noted that in vitro the neuroprotective
effects of testosterone or 17-estradiol treatments have also been
described [1,15]. Nevertheless, it seems that the action of nan-
drolone pre-treatment on the effect of MDMA on extracellular
5-HT changes dramatically along with the amount of nandrolone
administered. Whether this phenomenon results from increased
vulnerability of the neurons to acute stage of MDMA neurotox-
icity or to some other mechanism, remains to be elucidated.
The minor effect of sub-chronic nandrolone pre-treatment on
either DA or 5-HT metabolites compared with the transmitters
themselves suggests that nandrolones sites of action are not
monoamine oxidase enzymes (MAOs) that catalyze metabolic
conversion of DA to DOPAC, or 5-HT to 5-HIAA. Changes in
enzyme action would be reflected in changes in metabolic rates,
which were not observed. In addition, the concentrations of the
second metabolite of DA, HVA, paralleled those of DOPAC, as
expected.
In this study, amphetamine and MDMA produced a rapid
and long-lasting increase in LMA, which, especially in the
case of amphetamine, changed into stereotypic behavior. Sub-
199
chronic pre-treatment with nandrolone decreased amphetamine-
and MDMA-induced LMA. Amphetamine or MDMA-induced
scored behaviors, which predominantly consisted of stereotyp-
ies, were attenuated by both doses of nandrolone. These results
are concordant with the DA results of our microdialysis exper-
iments, as well as several early studies, in which testosterone
has been shown to attenuate amphetamine induced locomotor
activity and stereotyped behavior [5,16,46]. Correlation with
DA results is expected, because amphetamine-induced hyper-
locomotion is predominantly mediated by increased release of
DA in NAc [14].
Whether the nandrolone-induced changes in the neurochem-
ical and behavioral effects of amphetamine and MDMA were
mediated interaction of nandrolone with intracellular androgen
receptors is an interesting question. Indeed, such interaction
could lead to alterations in neuronal excitability and signal trans-
duction, which in turn could change organisms neurochemical
response to stimulant drug. However, because the density of
intracellular androgen receptors is shown to be low in the NAc
[48], it seems that if androgen receptors were involved, the
site of action should be somewhere outside the NAc. Neverthe-
less, although it seems clear that sub-chronic supraphysiological
treatment with nandrolone changes the neurochemical effects
of amphetamine and MDMA, the role of androgen receptors in
these changes remains to be elucidated.
Whether androgen receptors are involved or not, it is presum-
able that neurotransmitter systems other than dopaminergic or
serotonergic may also participate in mediating the effects of nan-
drolone observed here. Good candidates could be for example
GABAergic or glutamatergic neurotransmitter systems. AASs
has been reported to enhance and inhibit GABAA receptor bind-
ing [36,38], and alter the mRNA levels of glutamate NMDA
receptors in NAc [35].
It can also be speculated whether the ability of nandrolone
to modulate neurochemical effects of stimulant drugs is due to
nandrolone-induced changes in drug pharmacokinetics. Previ-
ous studies in rodents have shown that male and female rats
have comparable brain levels of MDMA after acute treatment
[12], suggesting that presence of physiological concentrations
of androgens do not have an effect on MDMA pharmacoki-
netics. According to our preliminary study, sub-chronic i.m.
pre-treatment with nandrolone either at doses of 5 or 20 mg/kg
caused no dramatic changes in brain tissue amphetamine con-
centration, the lowest concentration being measured for the
rat treated with arachinoid oil. If the decreased neurochemi-
cal or behavioral response to amphetamine was to be explained
by altered amphetamine pharmacokinetics, the brain concen-
tration of amphetamine should have been markedly lower in
nandrolone-treated rats than in arachinoid oil treated rats. In
contrast, MDMA brain concentrations in animals treated with
nandrolone were lower that of the one treated with arachinoid
oil; therefore, MDMA was also measured from blood samples
from 19 animals that were included in the neurochemical and
behavioral study. The means of the MDMA blood concentra-
tions were equal in every treatment group. Thus, based on these
results it can be concluded that it is unlikely that the reason
underlying the altered response to amphetamine or MDMA fol-
200 S. Kurling et al. / Behavioural Brain Research 189 (2008) 191201
lowing nandrolone pre-treatment is changed pharmacokinetics
of the stimulant drugs.
In conclusion, these data show that treatment with supraphys-
iological doses of the AAS, nandrolone decanoate, attenuates
reward-related neurochemical as well as behavioral effects
induced by amphetamine or MDMA dosing. Given that AASs
modulate functions of the brain reward systems, it is likely that in
addition to a desire to enhance ones exterior features or sporting
performance there are also neurochemical adaptations behind
prolonged misuse of AASs. The reduced activation of dopamin-
ergic neurons may correspond to the increased prevalence of
illicit drug usage among people who self-administer AAS and
that abusers of AAS may require larger doses of drugs to achieve
the desired effects. Whether these adaptations lead to increased
vulnerability to developing drug dependence, remains to be elu-
cidated. In addition, the mechanisms by which AASs modulate
functioning of dopaminergic and serotonergic neuronal systems
need further investigation.
Acknowledgements
The authors wish to thank the National Institute on Drug
Abuse (NIDA), for generously donating the MDMA HCl. This
research was supported in part by the Finnish Foundation for
Alcohol Studies.
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