Genes, Brain and Behavior (2009) 8: 161–173
Journal compilation # 2009 Blackwell Publishing Ltd/International Behavioural and Neural Genetics Society
Anabolic-androgenic steroid treatment induces
behavioral disinhibition and downregulation of
serotonin receptor messenger RNA in the prefrontal
cortex and amygdala of male mice
G. Ambar† and S. Chiavegatto*,†,‡,§
†
Department and Institute of Psychiatry, and ‡ Heart Institute
(InCor), University of São Paulo Medical School, and §Department
of Pharmacology, Biomedical Sciences Institute, University of
São Paulo, São Paulo, Brazil
*Corresponding author: S. Chiavegatto, MSc, PhD, Department
of Pharmacology, Biomedical Sciences Institute, University of
São Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900, São Paulo,
SP, Brazil. E-mail: schiaveg@usp.br
Nandrolone is an anabolic-androgenic steroid (AAS) that
is highly abused by individuals seeking enhanced phys-
ical strength or body appearance. Supraphysiological
doses of this synthetic testosterone derivative have been
associated with many physical and psychiatric adverse
effects, particularly episodes of impulsiveness and overt
aggressive behavior. As the neural mechanisms under-
lying AAS-induced behavioral disinhibition are
unknown, we investigated the status of serotonergic
system-related transcripts in several brain areas of mice
receiving prolonged nandrolone administration. Male
C57BL/6J mice received 15 mg/kg of nandrolone
decanoate subcutaneously once daily for 28 days, and
different sets of animals were used to investigate motor-
related and emotion-related behaviors or 5-HT-related
messenger RNA (mRNA) levels by real-time quantitative
polymerase chain reaction. AAS-injected mice had
increased body weight, were more active and displayed
anxious-like behaviors in novel environments. They ex-
hibited reduced immobility in the forced swim test, a high-
er probability of being aggressive and more readily
attacked opponents. AAS treatment substantially reduced
mRNA levels of most investigated postsynaptic 5-HT
receptors in the amygdala and prefrontal cortex. Interest-
ingly, the 5-HT1B mRNA level was further reduced in the
hippocampus and hypothalamus. There was no alteration
of 5-HT system transcript levels in the midbrain. In conclu-
sion, high doses of AAS nandrolone in male mice recapit-
ulate the behavioral disinhibition observed in abusers.
Furthermore, these high doses downregulate 5-HT recep-
tor mRNA levels in the amygdala and prefrontal cortex.
Our combined findings suggest these areas as critical sites
for AAS-induced effects and a possible role for the 5-HT1B
receptor in the observed behavioral disinhibition.
doi: 10.1111/j.1601-183X.2008.00458.x
# 2009 The Authors
Keywords: 5-HT1B receptor, aggression, anxiety, drug of
abuse, gene expression, hippocampus, hypothalamus, nan-
drolone, real-time PCR, serotonin
Received 27 May 2008, revised 3 September 2008, 18
October 2008, 21 October 2008, accepted for publication 27
October 2008
Anabolic-androgenic steroids (AASs) are synthetic com-
pounds derived from testosterone with therapeutic indications
to treat anabolic and androgenic disorders. Increasing world-
wide nonmedical use of AAS is prevalent in adolescents and
adults, typically in athletes or in individuals seeking physical
strength or enhanced appearance (Buckley et al. 1988; Lukas
1996; Pope & Katz 1988; Yesalis 1992; Yesalis et al. 1993,
1997). Abuse of AAS is based on supraphysiological doses
10–100 times higher than the therapeutic dose (Brower 1993;
Clark & Fast 1996) that have been associated with a wide
range of physical and psychiatric adverse effects. Early
behavioral effects include increased confidence, energy and
motivation accompanied by irritability and agitation (Midgley
et al. 2001; Pope & Katz 1988, 1994), whereas prolonged use
is usually associated with loss of inhibition and impulsive and
markedly aggressive behavior (Choi & Pope 1994; Corrigan
1996; Hall et al. 2005; Schulte et al. 1993). In fact, the most
evident reported personality disorders are increased episodes
of overt aggression or violent feelings and actions.
The neural mechanisms underlying this behavioral disinhi-
bition are unknown. Studies using rodents receiving supra-
physiological doses of AAS (>3 mg/kg/day, Clark & Fast
1996; Clark & Henderson 2003) have recapitulated some
behavioral side-effects, including elevated aggressive behav-
ior and disturbances in sexual behavior, but many results
remain controversial (reviewed in Clark & Henderson 2003).
The diversity of animals, strains, doses and regimen (i.e.
individual or different combinations of AAS) makes behavioral
data difficult to interpret.
Some recent studies have focused on brain molecular
mechanisms correlated with behavioral effects of high AAS
doses in rodents. Chronic use of AAS in mice has been shown
to induce dose-dependent, sex-dependent and age-dependent
changes in GABAA receptor subunit messenger RNA (mRNA)
levels in forebrain areas (McIntyre et al. 2002; reviewed in
Clark et al. 2006) suggesting involvement of GABAergic
inhibitory systems. Glutamatergic involvement has been
recently shown in adolescent AAS-treated hamsters through
161
Ambar and Chiavegatto
increased numbers of glutamate-expressing neurons as well
as GluR1 receptor expression in specific aggression-related
areas (Fischer et al. 2007). Additional studies by Melloni’s
group using adolescent male Syrian hamsters have provided
further evidence for participation of vasopressinergic and
serotonergic systems in AAS-induced aggression (reviewed
in Grimes et al. 2006). Specifically, significant increases in the
content of the neuropeptide arginine vasopressin (AVP) and in
its immunoreactive fiber density in the anterior hypothala-
mus, as well as elevated AVP V1A receptor binding in some
aggression-related areas, were observed (DeLeon et al. 2002;
Harrison et al. 2000). In this same model, a contribution of the
5-HT system has been suggested by decreased 5-HT inner-
vation and altered 5-HT1A and 5-HT1B receptor expression in
specific brain areas (Grimes & Melloni 2002, 2005; Ricci et al.
2006).
In light of the known role of 5-HT neurotransmission in
emotional behavior, particularly the participation of its dif-
ferent receptors in aggression (reviewed in Nelson &
Chiavegatto 2001), we investigated the emotional behaviors
of adult male mice receiving prolonged high doses of nan-
drolone decanoate, a highly abused type of AAS. Additionally,
we systematically quantified the mRNA levels of several
components in the 5-HT system (5-HT1A, 5-HT1B, 5-HT2A,
5-HT2C, 5-HT3A, 5-HT6 and 5-HT7 receptors, 5-HT transporter
and tryptophan hydroxylase) in different brain areas (pre-
frontal cortex, hippocampus, amygdala, hypothalamus and
midbrain) in a separate cohort of treated mice. This study thus
aimed to help elucidate, albeit in an indirect way, some
behavioral effects and critical neural substrates of anabolic
steroid abuse.
Materials and methods
Subjects and drugs
Male C57BL/6J mice from colonies bred and maintained in our
vivarium (Institute of Tropical Medicine, University of São Paulo
Medical School, Brazil) were isolated in standard polypropylene
cages at 3 months of age. They were randomly assigned to
steroid-treated or control groups, with no more than two littermates
included for each experimental condition. The steroid group received
subcutaneous injections of 15 mg/kg nandrolone decanoate (Deca-
durabolinâ, Organon, Brazil), and the control group received peanut
oil. This dose was chosen based on the prolonged dose–response
study performed in rats by Nyberg’s group (Lindblom et al. 2003).
The 15 mg/kg dose of nandrolone decanoate corresponds well to
heavy abuse of AAS (Pope & Katz 1988). AAS was given daily for
28 days in a volume of 10 ml/kg at around 0900 h. Animals were
kept on a 12-h light/dark cycle (lights on at 0700 h), with temperature
and humidity remaining fairly constant in closed, ventilated stands
(Alesco, São Paulo, Brazil). Autoclaved food (Purina Chow, São
Paulo, Brazil) and water were available ad libitum. Experiments were
carried out in accordance with the ‘Guidelines for the Care and Use
of Mammals in Neuroscience and Behavioral Research’ (Institute for
Laboratory Animal Research, USA), and the protocol was approved
by the Ethics Committee of the School of Medicine (University of
São Paulo, Brazil).
Behavioral assessments
Steroid and control animals (n ¼ 13 and n ¼ 16, respectively) were
weighed weekly. After 16 days of treatment, they were tested in four
162
behavioral paradigms, with a minimum 2-day recovery time between
each test (from 1400 to 1800 h). Test sequence and timing were
selected to minimize interference from previous behavioral proce-
dures, that is from less to more aversive test. Therefore, mice were
initially observed on the open field (OF), followed by elevated plus
maze (EPM), forced swim test (FST) and, finally, aggression test in the
resident–intruder model. During the total period of behavioral testing,
animals continued to receive steroid or vehicle injections in the
morning. Each test was performed alternately between groups, and
the equipment was cleaned with 5% ethanol before each test to avoid
odor cues. The experiments were videotaped overhead and scored
manually by a trained and blinded observer. The behavioral assess-
ment and killing were carried out in different sets of animals to
prevent manipulation effects on mRNA levels.
1 OF: a squared wooden 60 cm OF covered with white Formica
with 36 squares and 30 cm high walls (Bibancos et al. 2007) was used
to assess locomotor frequency (number of squares crossed), latency
to leave the center of the arena, rearing frequency (both front paws
elevated from the floor) and locomotor frequency in the 20 quadrants
adjacent to the walls for 5 min. Performance was assessed on the
16th day of treatment.
2 EPM: a wooden maze covered with black Formica with two
opposing open and closed arms (30
5 cm each and 15 cm high
walls in the closed arms) elevated 47 cm from the floor was used to
assess latency to leave the center (5 cm), entrance frequency in each
arm, time spent in each arm, transitions between the arms, frequency
of risk assessment (mice in the center of the maze investigating the
open arms without entering them with all four paws) and head-dipping
(according to Bibancos et al. 2007) for 5 min. Performance was
assessed on the 19th day of treatment.
3 FST: a glass cylinder of 27 cm in diameter and 40 cm deep was
filled with 30 cm of 248C water and used to determine frequency and
duration of immobility, that is the mouse floating motionless in the
water and only making movements necessary to keep its head above
water (modified from Porsolt et al. 1977a). Each mouse was tested
once for 6 min, and the duration of immobility during the last 4 min
was scored. Fresh water was replaced after each test. Performance
was assessed on the 25th day of treatment.
4 Resident–intruder: isolated steroid or control resident animals
were paired in their home cages with previously tested grouped-
housed adult docile male mice of the same strain. The following
behaviors displayed by residents during the 15-min encounter were
analyzed (according to Chiavegatto et al. 2001): latency to first attack
bite, number of bites, total duration of aggressive interactions and
number of animals showing aggression in each group. Performance
was assessed on the 28th day of treatment.
Brain and blood samples
Separate sets of same-aged animals were used for polymerase chain
reaction (PCR) analyses: n ¼ 10 each for steroid-treated and control
subjects. Animals were decapitated (alternating between treated and
control) on the 28th day of treatment, between 1400 and 1800 h.
Prefrontal cortex (anterior to corpus callosum), total hippocampus
(from both hemispheres), hypothalamus (whole hypothalamic area),
total amygdala (both left and right) and midbrain (central portion
without colliculi and substantia nigra) were carefully and immediately
dissected on ice by a trained researcher (Bibancos et al. 2007) based
on the mouse brain atlas (Paxinos & Franklin 2001) and snap frozen in
liquid nitrogen. Trunk blood was collected in heparinized microtubes,
centrifuged for 5 min at 12 000 g at 48C and the plasma separated. All
samples were stored at 808C until use.
Quantification of plasma androgenic steroids
Phenol/chloroform purified plasma samples were used in triplicate to
quantify circulating androgenic compounds using an enzyme immu-
noassay kit (Testosterone EIA kit from Cayman Chemical, Ann Arbor,
MI, USA) according to the manufacturer’s instructions. Cross-
reactivities for this EIA kit were 140% for 19-nortestosterone
Genes, Brain and Behavior (2009) 8: 161–173

(nandrolone), 100% for testosterone, 27.4% for 5a-dihydrotestoster-
one, 18.9% for 5b-dihydrotestosterone and less than 5% for all other
steroids. Consequently, the final plasma determination reflects a com-
bination of administered drug, basal testosterone and metabolite
levels. Quantification was performed using a Victor 1420 multi-label
counter (Wallac, Turku, Finland) at 405–420 nm.
RNA isolation and cDNA synthesis
Frozen brain samples were quickly immersed in TRIzol reagent
(Invitrogen, Carlsbad, CA, USA) and homogenized at maximum speed
(Polytron PT10/35-Brinkmann, Westbury, NY, USA) for 20 seconds.
Total RNA, isolated according to the manufacturer’s protocol, was
quantified and its purity assessed using a spectrophotometer (Nano-
Dropâ ND-1000, Thermo Fisher Scientific, Waltham, MA, USA). RNA
integrity was verified on 1% agarose gel stained with ethidium
bromide. Total RNA samples were treated with DNAse I enzyme
(Promega, Madison, WI, USA – 1 U/mg of RNA) for 30 min at 378C to
prevent residual DNA contamination. DNAse-treated total RNA (2 mg)
from both experimental groups was simultaneously reverse-tran-
scribed using oligo(dT) primers and SuperScript III reverse transcrip-
tase (Invitrogen, São Paulo, Brazil) in a final volume of 20 ml.
Primer design
Specific primers for serotonin 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT3A,
5-HT 6 and 5-HT 7 receptor genes, serotonin transporter 5-HTT
(Slc6a4), tryptophan hydroxylase-2 (Tph2) and the control genes
cyclophilin A (peptidylprolyl isomerase A: Ppia), hypoxanthine guanine
phosphoribosyl transferase 1 (Hprt1), glyceraldehyde-3- phosphate
dehydrogenase (Gapdh) and beta-actin (Actb) were previously de-
signed and are detailed elsewhere (Bibancos et al. 2007). Forward and
reverse primers were designed in different exons (with the exception
of the intronless genes Htr1a and Htr1b), and quality was further
confirmed using the NETPRIMER online tool (www.premierbiosoft.com/
netprimer/netprlaunch/netprlaunch.html). Primers were synthesized
by Invitrogen (Brazil) or IDT Technologies (Coralville, IA, USA).
Real-time quantitative PCR
Quantitative analysis of transcripts was carried out in a Rotor-Gene 3000
device (Corbett Research, Concord, Australia) using SYBR green.
Optimal conditions for cDNA and primer concentration as well as
amplification efficiency were obtained through five-point twofold dilution
curve analysis using RG-3000 (Corbett Research) software for each gene
and for different brain regions. As a result, each PCR reaction contained
12.5 ng of reverse-transcribed RNA, 200 nM of each specific primer,
SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA,
USA) and RNAse-free water to a 20 ml final volume. cDNA samples of
both groups were assayed in the same run, in triplicate, using 0.1 ml
microtubes. Samples without cDNA template (no template control), and
samples with RNA (no reverse transcription) were included as negative
controls in all experiments. Amplification parameters were 958C for
10 min, followed by 40 cycles at 958C for 15 seconds and 608C for
60 seconds, with fluorescence acquisition at the end of each step. A
melting step (dissociation curve) was performed following each run to
further confirm product specificity and absence of primer dimers. Real-
time data were analyzed using RG-3000 (Corbett Research) software
and an Excel spreadsheet. The relative amount of transcripts of interest
in different brain region for steroid and control groups was calculated
according to Vandesompele et al. (2002). Briefly, the arithmetic mean of
each transcript’s replicated cycling threshold (Ct) value was trans-
formed to a quantity relative to the sample with the highest level,
taking into account the amplification efficiency of each transcript. Real-
time PCR efficiencies for each reaction varied from 93% to 104%, and
the correlation coefficient was not lower than 0.96. Raw quantities
were subsequently normalized by dividing them by the geometric mean
of stable reference transcripts for each brain area, selected among
Gapdh, Actb, Hprt1 and Ppia genes according to the GENORM APPLET
version 3.4 (http://medgen.ugent.be/~jvdesomp/genorm/).
Genes, Brain and Behavior (2009) 8: 161–173
Anabolic steroid disinhibition and 5-HT receptor mRNA
Statistical analysis
The body weights of steroid and control mice during the 28 days of
treatment were compared by two-way analysis of variance for
repeated measures (factors: treatment and time) using SPSS 13.0.0
software (SSPS Inc., Chicago, IL, USA). Behavioral and mRNA data
were tested for normal distribution (Kolmogorov–Smirnov test), and
those with parametric distribution were compared using two-tailed
Student’s t-test, while nonparametric data were compared by Mann–
Whitney U-test (GRAPHPAD INSTATâ version 3.05, GraphPad Software,
San Diego, CA, USA). The frequency of animals showing aggressive
behavior in each group was compared using the chi-square test
(GRAPHPAD INSTAT version 3.05). Data were expressed as mean 
SEM or median and range. Differences were considered statistically
significant when P < 0.05.
Results
Effects of nandrolone administration on body weight
and circulating androgenic steroid levels in male mice
The body weight of both groups was similar at the beginning
of treatment (day 0). However, the steroid group showed
a gradual increase over the course of treatment
[F(1,27) ¼ 7.01, P < 0.05 for treatment; F(4,140) ¼ 38.20, P <
0.01 for time and F(4,140) ¼ 13.89, P < 0.01 for interaction;
Fig. 1a]. Plasma androgenic steroid levels on the 28th day of
nandrolone treatment (5–8 h after the last drug injection)
were 6.7 times higher than those of control mice (P < 0.01;
Fig. 1b).
Effects of nandrolone administration on OF activity
Total OF locomotor frequency was increased in the steroid
group (control, n ¼ 16: 127.63  11.46 vs. steroid, n ¼ 13:
173.46  12.51, P < 0.05). Increased spontaneous motor
activity occurred in the quadrants along the walls (P < 0.05)
but not in the central quadrants (P > 0.05) (Fig. 2a). The
higher rearing frequency of male mice receiving nandrolone
almost reached statistical significance (P ¼ 0.06) (Fig. 2a).
Latency to leave the central area did not differ between
groups (control, n ¼ 16: 31.64  5.21 seconds vs. steroid,
n ¼ 13: 22.22  3.28 seconds, P > 0.05).
Effects of nandrolone administration on anxiety/
fear-like behaviors
Anxiety-related behaviors were investigated using both EPM
and OF apparatus. The time spent in the most aversive areas
was reduced for the steroid group. In the EPM, time spent in
the open areas (open arms and central area) was reduced in
the steroid group (P < 0.05) (Fig. 2b), while maintaining
similar entrance frequency in the open arms (control, n ¼ 16:
2.06  0.51 vs. steroid, n ¼ 13: 1.69  0.43, P > 0.05) and
transitions between arms (control: 16.06  0.98 vs. steroid:
18.08  1.24, P > 0.05). In the OF, mice in the steroid group
spent less time in quadrants of the central area compared
with those in the control group (P < 0.05) (Fig. 2b). Other
behavioral parameters related to anxiety/fear included
reduced latency to escape from the EPM central area (P <
0.05) (Fig. 3c) and increased frequency of risk assessments
in the steroid group (P < 0.01) (Fig. 3c). The frequency of
163
Ambar and Chiavegatto
Figure 1: Weekly body weight of male mice receiving nandrolone and plasma levels of androgen steroids on the 28th day of
daily treatment. (a) The steroid group (n ¼ 13) showed a significant increase in body weight (g) starting on the 14th day that persisted
throughout the treatment when compared with the control group (n ¼ 16) (P < 0.01). (b) Androgen steroid quantification in the plasma
after 28 days of nandrolone injections. The immunoassay results do not discriminate between nandrolone, testosterone and their
metabolites (** P < 0.01). Data are expressed as mean and SEM.
head-dipping did not differ between groups (control, n ¼ 16:
13.00  1.49 vs. steroid, n ¼ 13: 11.77  1.24, P > 0.05).
Effects of nandrolone administration on FST
The duration of immobility in the last 4 min of the FST was
reduced in the steroid group when compared with the
controls (P < 0.05) (Fig. 3).
Effects of nandrolone administration on aggressive
behavior
Animals receiving nandrolone showed more aggressive be-
haviors in the resident–intruder paradigm (attacks with bites,
pursuing and aggressive interactions) than those receiving
the vehicle (P < 0.05) (Fig. 4). The steroid group had shorter
latency to the first attack (P < 0.05) (Fig. 4), but the number
of attack bites did not differ during the 15-min testing interval
[control: 0 (0–52), n ¼ 16 vs. steroid: 8.5 (0–46), n ¼ 12;
Mann–Whitney U-test, P ¼ 0.08].
Effects of nandrolone administration on 5-HT receptor
mRNA levels in the prefrontal cortex
Transcript levels of 5-HT1A, 5-HT1B, 5-HT2A and 5-HT7 recep-
tors were statistically reduced in the prefrontal cortex of mice
receiving steroids when compared with the control group
(Fig. 5). The decrease in mRNA levels was 62.9% for 5-
HT1A (P < 0.01), 58.8% for 5-HT1B (P < 0.01), 52.9% for
5-HT2A (P < 0.01) and 42.4% for 5-HT7 (P < 0.05). The
remaining 5-HT receptor transcripts in this brain area, 5-HT2C,
5-HT3A and 5-HT6, did not vary across groups (P > 0.05)
(Fig. 5). The control transcript used to normalize 5-HT recep-
tor mRNA levels in the prefrontal cortex was Actb (control:
0.40  0.08 vs. steroid: 0.25  0.03, n ¼ 10 each group,
P > 0.05). Levels of Ppia, Hprt1 and Gapdh mRNAs were
statistically reduced in the steroid group in relation to controls
164
(Ppia: 0.61  0.08 vs. 0.40  0.04, P < 0.05; Hprt1:
0.51  0.07 vs. 0.25  0.04, P < 0.01; Gapdh: 0.51  0.08
vs. 0.29  0.03, P < 0.05, n ¼ 10 for each group, control vs.
steroid, respectively). Consequently, these mRNAs were not
used for normalization in this brain area.
Effects of nandrolone administration on 5-HT receptor
mRNA levels in the hippocampus
5-HT1B was the only 5-HT receptor transcript whose level was
statistically different in the hippocampus of steroid mice with
respect to the control group; it showed a reduction of
36.6% (P < 0.05) (Fig. 6). In the hippocampus, Hprt1 and
Actb mRNAs levels were used to normalize 5-HT receptor
transcripts because of their stable levels (M ¼ 0.67, GENORM
APPLET). In this structure, all four control genes had similar
mRNA levels across groups (Ppia: 0.47  0.07 vs.
0.56  0.09; Hprt1: 0.37  0.07 vs. 0.49  0.07; Actb:
0.33  0.09 vs. 0.36  0.07; Gapdh: 0.36  0.09 vs.
0.31  0.07, n ¼ 10 for each group, control vs. steroid,
respectively, P > 0.05).
Effects of nandrolone administration on 5-HT receptor
mRNA levels in the amygdala
Transcript levels of 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT6 and
5-HT7 receptors were statistically reduced in the amygdala of
mice receiving nandrolone when compared with the control
group (Fig. 7), with decreases of 48.4% for 5-HT1A (P <
0.01), 46.0% for 5-HT1B (P < 0.05), 36.5% for 5-HT2A
(P < 0.05), 66.4% for 5-HT2C (P < 0.01), 42.4% for 5-HT6
(P < 0.05) and 61.0% for 5-HT7 (P < 0.01). The remaining
5-HT transcript quantified in this brain area, 5-HT3A, did not
vary between groups (P > 0.05) (Fig. 7). In the amygdala,
Ppia and Hprt1 mRNAs were used to normalize 5-HT receptor
transcripts because of their stability (M ¼ 0.64, GENORM
APPLET). Gapdh mRNA levels were reduced in the steroid
Genes, Brain and Behavior (2009) 8: 161–173
 Anabolic steroid disinhibition and 5-HT receptor mRNA

Figure 2: OF activity and anxiety/fear-related

behaviors of male mice with prolonged nan-
drolone administration. (a) The locomotor fre-
quency in 5 min was significantly higher along
the walls but not in central quadrants in the
steroid group (n ¼ 13), whereas rearing fre-
quency almost reached statistical significance
when compared with the control group (n ¼ 16).
(b) Time spent in aversive areas of EPM and OF
was significantly reduced in the steroid group
(n ¼ 13) when compared with controls (n ¼ 16),
although transitions between arms of EPM were
similar. (c) Reduced latency to escape the central
EPM area and increased frequency of risk as-
sessments in the steroid group. Data are ex-
pressed as mean and SEM. * P < 0.05 and
** P < 0.01.

group (0.66  0.09 vs. 0.39  0.09, n ¼ 10 each, P < 0.05), 0.46  0.08 vs. 0.46  0.11; Actin: 0.63  0.09 vs.
but levels of the other three controls tested were similar 0.34  0.13, P > 0.05, n ¼ 10 in each group, control vs.
across groups (Ppia: 0.47  0.07 vs. 0.42  0.09; Hprt1: steroid, respectively).

Genes, Brain and Behavior (2009) 8: 161–173 165

Ambar and Chiavegatto
Figure 3: Immobility duration in the FST paradigm after
prolonged nandrolone administration. Immobility time was
significantly decreased in the last 4 min of the FST in the steroid
group (n ¼ 13) when compared with controls (n ¼ 16). Data are
expressed as mean and SEM. * P < 0.05.
Effects of nandrolone administration on 5-HT receptor
mRNA levels in the hypothalamus
The level of 5-HT 1B receptor transcripts was reduced
(51.5%, P < 0.05) and that of 5-HT6 receptor mRNA was
increased (þ44.3%, P < 0.05) in the hypothalamus of mice in
the steroid group when compared with the control group
(Fig. 8). The other 5-HT receptor transcript levels were similar
across groups (P > 0.05) (Fig. 8). In the hypothalamus, Hprt1
and Gapdh showed the most stable levels (M ¼ 0.56, GENORM
APPLET) and were thus used for the normalization of transcripts
encoding 5-HT receptors. Levels of the four control genes
tested did not vary across groups [Ppia: 0.39  0.12 (n ¼ 9)
vs. 0.37  0.05 (n ¼ 7); Hprt1: 0.44  0.11 (n ¼ 9)
vs. 0.56  0.10 (n ¼ 7); Actb: 0.36  0.11 (n ¼ 9) vs.
0.45  0.09 (n ¼ 7); Gapdh: 0.40  0.11 (n ¼ 9) vs.
Figure 4: Aggressive behavior of male mice after prolonged
nandrolone administration. More animals receiving steroids
displayed aggressive behavior in the resident–intruder paradigm.
Mice in the steroid group (n ¼ 13) were significantly faster to
attack their opponents when compared with the control group
(n ¼ 16). Data are expressed as mean and SEM. * P < 0.05.
166
Figure 5: 5-HT receptor transcripts in the prefrontal cortex of
male mice after prolonged nandrolone administration. Levels
of transcripts for 5-HT1A, 5-HT1B, 5-HT2A and 5-HT7 receptors
were significantly reduced (decreases ranged from 42.4% to
62.9%) in the steroid group (n ¼ 10) when compared with
controls (n ¼ 10). Transcripts in the prefrontal cortex were
normalized against Actb mRNA levels. Data are expressed as
mean and SEM. * P < 0.05 and ** P < 0.01.
0.38  0.09 (n ¼ 7), control vs. steroid, respectively,
P > 0.05].
Effects of nandrolone administration on 5-HT-related
mRNA levels in the midbrain
The level of transcripts for all 5-HT receptors tested, as well
as for the 5-HTT and Tph2 genes, did not vary across groups in
the midbrain area (Table 1). In this region, Hprt1 and Gapdh
showed the most stable levels (M ¼ 0.40, GENORM APPLET) and
were consequently used for normalization of 5-HT-related
Figure 6: 5-HT receptor transcripts in the hippocampus of
male mice after prolonged nandrolone administration. The
5-HT1B receptor transcript was the only altered transcript (36.6%)
in the hippocampus of mice in the steroid group (n ¼ 10) when
compared with controls (n ¼ 10). Transcripts in the hippocampus
were normalized against the geometric mean of Actb and Hprt1
mRNA levels. Data are expressed as mean and SEM. *P < 0.05.
Genes, Brain and Behavior (2009) 8: 161–173

Figure 7: 5-HT receptor transcripts in the amygdala of male
mice after prolonged nandrolone administration. All 5-HT
receptor transcripts, except for the 5-HT3A receptor, were signi-
ficantly decreased in the amygdala of mice in the steroid group
(n ¼ 10) when compared with controls (n ¼ 10). Reductions
ranged from 36.5% to 66.4%. Transcripts in the amygdala
were normalized against the geometric mean of Hprt1 and Ppia
mRNA levels. Data are expressed as mean and SEM. *P < 0.05
and **P < 0.01.
transcripts. Ppia mRNA level was decreased in the steroid
group (control: 0.63  0.09 vs. steroid: 0.28  0.05, n ¼ 8
each, P < 0.01), but the levels of the other tested control
genes were similar for both groups (Hprt1: 0.70  0.09 vs.
0.49  0.09; Actb: 0.53  0.09 vs. 0.44  0.13 and Gapdh
0.67  0.07 vs. 0.44  0.10, n ¼ 8 per group, control vs.
steroid, respectively, P > 0.05).
Discussion
Prolonged administration of a supraphysiological AAS nan-
drolone dose to adult male mice was able to recapitulate
several characteristics observed in individuals who illicitly use
synthetic analogs of testosterone. Abuse of these com-
pounds is related to their anabolic properties, that is gain in
lean body mass (Bahrke & Yesalis 2004; Bhasin et al. 1996;
van Marken Lichtenbelt et al. 2004). Accordingly, we
observed increased body weight in our mice starting as soon
as the second week of nandrolone administration that per-
sisted throughout the 28-day course of AAS injections.
Nandrolone is one of the most abused types of AAS because
of its decreased androgenic activity (Clark & Henderson 2003;
NIDA 2001; Shahidi 2001), although it is not devoid of adverse
Table 1: 5-HT-related transcripts in the midbrain of male mice after prolonged nandrolone administration
5-HT receptors
Groups 1A 1B 2A 2C
Control 0.73  0.17 1.03  0.12 0.80  0.09 0.95  0.11 0.70  0.13 1.13  0.09 0.88  0.12 0.74  0.11 0.90  0.12
Steroid 0.79  0.13 1.13  0.08 0.96  0.08 1.07  0.14 0.69  0.11 1.03  0.10 0.79  0.09 0.85  0.15 1.02  0.14
Transcripts in the midbrain were normalized by the geometric mean of Hprt1 and Gapdh mRNA levels. Data are expressed as mean  SEM.
n ¼ 8, for both groups.
Genes, Brain and Behavior (2009) 8: 161–173
Anabolic steroid disinhibition and 5-HT receptor mRNA
Figure 8: 5-HT receptor transcripts in the hypothalamus of
male mice after prolonged nandrolone administration. The
5-HT1B receptor transcript level was significantly reduced
(51.5%), whereas the 5-HT6 receptor transcript level was
elevated (þ44.3%) in the hypothalamus of mice in the steroid
group (n ¼ 10) when compared with the control group (n ¼ 10).
The mRNA levels of the other receptors were similar between the
groups (P > 0.05). Transcripts in the hypothalamus were normal-
ized against the geometric mean of Hprt1 and Gapdh mRNA
levels. Data are expressed as mean and SEM. *P < 0.05.
effects. Users have reported neurobehavioral changes, such
as euphoria and supra-aggression (Parrott et al. 1994; Pope
et al. 2000; Su et al. 1993); analogous behaviors were also
observed in the present study in mice. Because of the
likelihood of 5-HT involvement in these behavioral manifes-
tations, we hypothesized that 5-HT-related mRNA levels
would be altered in this system. We were able to detect
several changes in 5-HT receptor transcript levels in distinct
aggression-associated brain areas of mice given an AAS
treatment; such treatment also elicited enhanced aggression
in a separate cohort of animals.
Behavior
Male adult C57BL/6J mice receiving daily injections of a high
dose of nandrolone decanoate were more active when evalu-
ated for 5 min in the OF. Higher motor activity was represented
by increased locomotor frequency and a tendency toward
increased rearing. Locomotor stimulation in mice is observed
after administration of addictive drugs and is suggested to be
related to the euphoric effect induced by these drugs in humans
(Bergstrom et al. 2003). This stimulatory behavior in a novel
3A 6 7 5-HTT Tph2
167
Ambar and Chiavegatto
environment occurred only in quadrants adjacent to the walls,
which are the most protected areas for rodents. Cautious
behavior was also reflected in EPM exploration. In this
apparatus, mice receiving nandrolone spent less time in the
aversive areas (center and open arms), escaped faster from
the central area and also displayed increased frequency of risk
assessment before entering the aversive areas. Altogether,
these results suggest elevated anxiety or fear-like behaviors in
addition to AAS-induced motor stimulation.
In the FST, rodents are placed in a restricted container of
water from which they cannot escape. After initial vigorous
activity, they adopt a characteristic immobile posture sugges-
tive of behavioral despair or a passive coping strategy; this
behavior is selectively inhibited by several treatments effective
for depression (Porsolt et al. 1977a,b). In the present study,
mice receiving nandrolone exhibited reduced immobility time
in this test when compared with the control group. This result
is consistent with the hyperactive phenotype observed in the
OF and thus suggests that reduced immobility in the FST
could result from general arousal rather than delayed onset of
inactivity. In fact, psychostimulants can also reduce the
immobility of rodents in the FST because of a pronounced
increase in motor activity, introducing a caveat to the inter-
pretation of this test (Porsolt et al. 1977a). However, other
groups have also found no correlation between OF and FST
activities, suggesting that activity in each test is related to
different phenomena (Alonso et al. 1991). In support of this
concept, a previous study in our laboratory showed that after
prolonged social isolation after weaning, aggressive C57BL/
6J male mice showed hyperactivity in novel environments
(Bibancos et al. 2007) but no difference in immobility time in
the FST compared with group-housed controls (unpublished
data). These data further suggest that different or additional
neural mechanisms could participate in the duration of FST
immobility. Therefore, nandrolone-mediated reduction of
mouse immobility in the FST should be further explored,
especially because antidepressant effects of high AAS doses
have been reported in humans (Uzych 1992).
Mice receiving nandrolone initiated attacks against in-
truders faster than mice receiving vehicle. Additionally, 75%
of mice in the steroid group displayed aggressive behavior in
the 15-min observational interval, whereas only 33% of the
control group did so. Based on Brunner and Hen’s (1997)
characterization of the failure to inhibit behavior as a motor
aspect of impulsivity (reviewed in Arce & Santisteban 2006),
we suggest that nandrolone treatment increased the impul-
sive behavior of these animals. They reacted more quickly to
both the social stimulus (encounter with a conspecific) and
the fear-induced stimulus (central area of the EPM). This
impulsive, reactive type of aggression is also observed in
individuals abusing AAS (Hall et al. 2005) and is in agreement
with the proposed ability of androgen steroids to elevate
sensitivity to external stimuli and lower the threshold for ag-
gression and dominance in response to social threats (Breuer
et al. 2001; Hermans et al. 2008; McGinnis et al. 2002).
Quantification of mRNA transcripts
Affective behavioral effects of high AAS doses in humans
might partly be explained by disturbances in 5-HT neurotrans-
168
mission. AAS has different effects that are determined by
location and cell type; many are believed to be mediated by
hormonal binding to an intracellular protein with subsequent
translocation of the resulting complex to binding sites on
chromatin to modulate gene transcription and subsequent
mRNA synthesis (Spelsberg et al. 1989). Evidence that has
accumulated over the past two decades has suggested
additional nongenomic actions of androgens and other ste-
roids (reviewed in Foradori et al. 2008). These are mediated
by non-classical receptors, which are implicated in rapid
effects on cellular functions and can be found in neuronal
and glial processes (Sarkey et al. 2008). Nandrolone (19-
nortestosterone) directly activates androgen receptors and
through its aromatization to 17b-estradiol (although only
20% as efficiently as testosterone) (Ryan 1959) has addi-
tional effects at brain estrogen receptors through estrogenic
metabolites. All these effects likely occurred in the present
study because of the daily and prolonged administration of
high doses of AAS. The impact of such dosing is reflected by
the 6.7-fold increase in circulating androgen steroid levels in
comparison to controls on the 28th day of treatment. We
could not identify any steroid-response elements in the gene
sequences encoding 5-HTT, Tph2 or the 5-HT receptors that
might predict a direct effect of AAS (or its estrogenic
metabolites) on their transcription. Therefore, we speculate
that indirect AAS modulation, either by non-classical steroid
receptors or by a classical effect on steroid-response ele-
ments in other genes, ultimately affects the mRNA levels of
genes related to 5-HT neurotransmission.
Serotonergic axons primarily originate from neuronal cell
bodies in the dorsal and median raphe nuclei in the midbrain
and project to both rostral and caudal areas of the brain
(Aghajanian et al. 1967; Azmitia & Whitaker-Azmitia 1991).
We selected several brain areas related to emotional behav-
iors that are enriched in 5-HT receptors and steroid receptors.
Transcripts encoding 5-HT receptors obtained from the fore-
brain regions (prefrontal cortex, hippocampus, amygdala and
hypothalamus) likely originated from nonserotonergic neu-
rons with postsynaptic 5-HT receptors (Fig. 9). Transcripts
encoding 5-HT receptors, 5-HT transporter or 5-HT synthetic
enzyme harvested from the midbrain area were likely derived
from serotonergic neuronal somata, but additional nonsero-
tonergic neurons from local circuits cannot be excluded.
5-HT receptor transcripts in the amygdala
Converging evidence from animal and human studies impli-
cates the amygdala as a critical area of emotional regulation
and aggression (Coccaro et al. 2007; Davidson et al. 2000;
Nelson & Trainor 2007). Amygdala hyperexcitability has been
suggested to partly contribute to the neural basis of fear and
anxiety disorders, impulse control and aggressive behaviors
(reviewed in Keele 2005). The amygdala of rodents exhibits
strong immunoreactivity for androgen and estrogen receptors
(Apostolinas et al. 1999; Mitra et al. 2003; Sar et al. 1990;
Sarkey et al. 2008). High dosing of nandrolone for 2 weeks in
guinea pigs increased the number of c-Fos-positive neurons
in the central amygdala, showing activation of this brain area
by AAS (Johansson-Steensland et al. 2002). In our study,
Genes, Brain and Behavior (2009) 8: 161–173

Figure 9: Schematic drawing of a 5-HT neu-
ron projection in a sagittal view of the mouse
brain. Represented are the presynaptic and post-
synaptic areas investigated in the AAS-treated
mice. Note the magnification of a synaptic cleft
from the prefrontal cortex (terminal ending of a 5-
HT neuron and a postsynaptic area of a non 5-HT
neuron showing 5-HT heteroreceptors).
prolonged administration of nandrolone induced a marked and
extensive downregulation of 5-HT receptor transcripts in the
amygdala. The levels of all 5-HT receptor transcripts, with the
exception of 5-HT3A, were significantly decreased by 37%
to 66% in steroid-treated mice when compared with the
control group. In agreement with our findings, downregula-
tion of 5-HT2 receptors was reported in the amygdala after
treatment with high doses of nandrolone in binding studies
with male rats (Kindlundh et al. 2003), and a decrease in
5-HT1B immunoreactive puncta was found in both the central
and the medial amygdala of AAS-treated aggressive male
hamsters (Grimes & Melloni 2005).
5-HT in the amygdala has been shown to activate GABAer-
gic inhibitory interneurons, resulting in a net inhibitory tone on
neuronal excitability (Rainnie 1999). This suggests that 5-HT
functions as a braking mechanism to limit neuronal excitability
(Keele 2005). The specific participation of different 5-HT
receptor subtypes expressed in the amygdala on this inhibi-
tory modulation is unknown. It is possible that nandrolone-
mediated downregulation of 5-HT receptor mRNA, and
potentially of 5-HT receptor protein synthesis, may result in
increased excitability in the amygdala, which in turn could
underlie the behavioral responses seen with prolonged use of
AAS. Accordingly, it has been recently shown that high doses
of AAS in pubertal male rats significantly increased spine
densities on neurons in regions of the amygdala, suggesting
increased formation of excitatory synapses in these areas
(Cunningham et al. 2007).
Interestingly, the 5-HT3 receptor, the only member of the
5-HT receptor family that belongs to the class of ionotropic
receptors and mediates 5-HT fast excitatory responses
(Sugita et al. 1992), was the only subtype in the present
study whose level was not affected by nandrolone. The
5-HT3A receptor has previously been associated with reactiv-
ity to environmental stimulation using pharmacological and
genetic manipulation techniques in mice (Bhatnagar et al.
2004; Gao & Cutler 1992; Kelley et al. 2003; Olivier et al.
2000). However, our data do not support mRNA level
participation of this receptor in the effects of nandrolone in
the amygdala.
Genes, Brain and Behavior (2009) 8: 161–173
Anabolic steroid disinhibition and 5-HT receptor mRNA
5-HT receptor transcripts in the prefrontal cortex
A role for the prefrontal cortex in behavioral inhibition, general
activity, attention and emotion is supported by lesion and
imaging studies (Fuster 2001). Recently, regional studies in
rats concluded that the cerebral cortex is the main forebrain
target for androgen action because it contains extensive
number of cells expressing androgen receptors (DonCarlos
et al. 2006). Accordingly, prolonged high dosage of nandro-
lone in guinea pigs activates the frontal cortex as shown by
increased Fos-related antigen-containing neurons (Johansson-
Steensland et al. 2002). In the present study, nandrolone
induced a substantial and significant decrease of 5-HT1A,
5-HT1B, 5-HT2A and 5-HT7 mRNA levels ranging from 42%
to 63% in the prefrontal cortex of male mice when compared
with vehicle-treated mice. In accord with our findings, 5-HT2
receptor density was significantly reduced in the frontal cortex
of male rats receiving similar doses of nandrolone for 2 weeks
(Kindlundh et al. 2003).
Interestingly, we have recently shown that increased
aggressive behavior of male mice generated by different
types of stimuli occurs concomitantly with downregulation
of most 5-HT receptor transcripts, particularly in the prefrontal
cortical region. While male mice socially isolated from wean-
ing displayed a hyperactive and aggressive phenotype paral-
leled by an overall reduction of 5-HT receptor mRNA levels in
the prefrontal cortex (Bibancos et al. 2007), mice exhibiting
escalated aggression after consumption of alcohol showed
a similar decrease in 5-HT receptor transcripts, with the
exception of 5-HT3A subtype (Chiavegatto et al. 2007).
Collectively, these data suggest a strong correlation between
aggressive behavior and perturbations of 5-HT receptor
mRNA levels in the prefrontal cortex.
5-HT receptor transcripts in the hippocampus
Ultrastructural analysis has shown androgen receptor immu-
noreactivity at several extranuclear sites in all hippocampal
subregions (Tabori et al. 2005). Estrogen receptors are
also present in the hippocampal formation (Spencer et al.
2008). Despite the presence of steroid receptors and the
169
Ambar and Chiavegatto
well-established role of the hippocampal formation in mem-
ory processes, chronic AAS treatments in male rats did not
induce deficits of spatial memory (Clark et al. 1995; Smith
et al. 1996) or hippocampal cell survival (Clark et al. 1995).
This could suggest that this brain region is less vulnerable to
perturbations induced by AAS. However, a role of the
hippocampus in anxiety independent of its roles in learning
and memory has also been proposed (Engin & Treit 2007).
In line with this reasoning, 5-HT1B was the only 5-HT
receptor subtype with altered mRNA content in the hippo-
campus in the present study. Prolonged administration of
nandrolone downregulated 5-HT1B receptor mRNA in male
mice to the order of 37% compared with the control group.
High nandrolone doses in guinea pigs have also resulted in
a marked decrease of 5-HT1B receptor density in the CA1
region of the hippocampus (Kindlundh et al. 2003). Although
the significance of this finding is unknown, it has been shown
that CA1 pyramidal neurons containing 5-HT1B mRNA project
primarily to the dorsal subiculum (Aı̈t Amara et al. 1995),
which is considered to be their major output pathway from
the hippocampus (Swanson & Cowan 1977). In the subicu-
lum, these presynaptic 5-HT1B receptors suppress glutamate
release from hippocampus, thereby modulating subicular
functions (Boeijinga & Boddeke 1993, 1996). Downregulation
of 5-HT1B receptor mRNA, and possibly 5-HT1B receptor
protein, by nandrolone treatment, would consequently re-
duce the inhibitory control of this area. Gray and McNaughton
(2000; reviewed in McNaughton 2006) suggested roles for
the hippocampus and subiculum in behavioral inhibition,
increased negative affective bias, increased exploration and
risk assessment. Our behavioral results in mice receiving high
nandrolone doses also suggest the participation of subiculum
activation in this anxious-like behavior. This is an interesting
hypothesis that deserves further investigation.
5-HT receptor transcripts in the hypothalamus
The hypothalamus is a classical neuroendocrine control
region that is highly sensitive to circulating levels of steroids.
Nuclear and cytoplasmic immunoreactivity for the androgen
receptor has been found in several nuclei of the hypothala-
mus in humans (Fernández-Guasti et al 2000) and rodents
(Sar et al. 1990). Mice receiving high doses of nandrolone
showed a 52% downregulation of 5-HT1B mRNA levels in the
hypothalamus when compared with control animals. In this
brain area, 5-HT6 receptor mRNA was also significantly
upregulated. The physiological implication of this finding is
not known because it has been shown that the brain
distribution of 5-HT6 receptor mRNA and protein, as well as
5-HT6 receptor pharmacology, is markedly different between
mice and rats or humans (Hirst et al. 2003). The uniqueness of
the 5-HT6 receptor in mouse brains and its modulation by AAS
in the hypothalamus warrant future studies.
5-HT-related transcripts in the midbrain
Levels of all 5-HT-related transcripts investigated in the mid-
brain area were quantitatively similar in mice receiving
prolonged nandrolone when compared with control mice.
170
The 5-HT synthetic enzyme, the 5-HT transporter and the
5-HT1A and 5-HT1B autoreceptors are part of the modulatory
machinery of 5-HT neurons; this machinery functions as
a feedback mechanism to control the activity of these cells.
The observed absence of changes in mRNA levels suggests
that effects of nandrolone on 5-HT transmission occur
postsynaptically.
Reduced 5-HT1B mRNA levels in 5-HT postsynaptic
brain areas
Our results suggest an important participation of the 5-HT1B
subtype in the AAS-mediated behavioral phenotype. Because
transcripts encoding 5-HT receptors obtained from the fore-
brain areas mostly encode 5-HT heteroreceptors, that is
receptors found in nonserotonergic neural cells, our results
implicate a role for postsynaptic 5-HT1B receptors in behav-
ioral disinhibition. Accordingly, 5-HT1B receptors have already
been linked to aggressive behaviors in rodents by genetic and
pharmacological studies. Increased impulsivity and aggres-
sive behavior has been found in mice in which the gene
encoding the 5-HT1B receptor was deleted by homologous
recombination (Saudou et al. 1994), and different 5-HT1B
receptor agonists reduce or abolish aggressive behavior in
rodents (Chiavegatto et al. 2001; De Almeida et al. 2006; Fish
et al. 1999; Olivier et al. 1995). Therefore, it has been
postulated that postsynaptic 5-HT1B heteroreceptors directly
influence the executive and consummatory phases of ago-
nistic behavior (Olivier & van Oorschot 2005).
Implications of nandrolone-induced changes in
mRNA levels
An important and novel result of this study is the site-specific
alteration (mostly downregulation) of the mRNA levels of
several 5-HT receptors by prolonged administration of high
doses of nandrolone. As mentioned before, we could not
identify steroid-response elements in the gene sequences of
these 5-HT receptors, suggesting an indirect or non-classical
effect of the AAS. The levels of mRNA quantified in this study
represent steady-state levels and thus may reflect altered
mRNA transcription, mRNA stability or both. In any case,
these effects may arise from signaling pathways that are
separate from the classical nuclear hormone receptor path-
ways, but at some point may also affect gene transcription
products. These preliminary and provocative findings broaden
the potential impact of AAS on cellular function and empha-
size the regional specificity of these effects on the brain.
It would be of interest to investigate the impact of AAS
administration on mRNA levels of components of different
neurotransmitter/neurosignaling systems. Finally, if 5-HT
receptor protein levels show similar changes to those
observed in the present study, such modulation might help
to explain the aggressive and anxious-like phenotype induced
by nandrolone in male mice.
Taken together, these data support the hypothesis that the
outcome of AAS abuse is not limited to desired anabolic
effects; it may also alter neurotransmission circuitry through-
out the central nervous system.
Genes, Brain and Behavior (2009) 8: 161–173

Conclusions
This study showed behavioral alterations as well as distur-
bances in mRNA levels of 5-HT receptors in different brain
areas after prolonged high-dose administration of nandrolone.
This is the first comprehensive investigation of the effects of
supraphysiological dosing AAS on 5-HT receptor mRNA levels
in male mouse brain regions implicated in emotion regulation.
In addition to increased body mass, male mice receiving
nandrolone had increased motor activity and anxious-like
behavior in novel situations. Importantly, they were more
aggressive and more impulsive to initiate attacks than mice
receiving vehicle. Regarding the steady-state levels of 5-HT
receptor transcripts, the most affected areas were the
amygdala and prefrontal cortex. In line with the behavioral
disinhibition, the substantial and extensive decrease of 5-HT
receptor subtype transcript levels suggests reduced 5-HT-
mediated inhibition of neuronal activity. Altered levels of
5-HT-system-related transcripts were not observed in pre-
synaptic areas, that is, 5-HT neuronal bodies. Reduced levels
of 5-HT1B receptor mRNA in all four 5-HT postsynaptic brain
sites strengthens the proposed role for 5-HT1B heteroreceptors
in aggressive and impulsive behaviors.
Although we cannot exclude posttranscriptional effects or
different modulatory actions at the protein level on 5-HT
neurotransmission, our findings in mice support the psychi-
atric side-effects observed in humans abusing AAS and
further shed light on the molecular mechanisms underlying
these adverse effects.
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Acknowledgments
We are grateful to Dr José Eduardo Krieger for laboratory
facilities and to Dr Hiro Goto for the vivarium. This study was
supported by grants from the São Paulo State Foundation for
Research Support (FAPESP: 06/06904-5). G.A. was a recipient of
CAPES (Ministry of Education Council for Professional Develop-
ment) master’s fellowship and S.C. is a research scholar of CNPq
(The National Council for Scientific and Technological Develop-
ment), Brazil.
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