Behavioural Brain Research 231 (2012) 181–186
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Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
GABA system changes in methylphenidate sensitized female rats
L. Freese a,b,∗ , E.J. Muller a , M.F. Souza a , N.S. Couto-Pereira a , C.F. Tosca a , M. Ferigolo b , H.M.T. Barros a
a
b
Division of Pharmacology, Basic Health Sciences Department, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil
Universidade Federal de Ciências da Saúde de Porto Alegre/National Services of Orientation and Information on the Prevention of Drug Use – VIVAVOZ, Brazil
a r t i c l e
i n f o
Article history:
Received 13 August 2011
Received in revised form 6 March 2012
Accepted 10 March 2012
Available online 20 March 2012
a b s t r a c t
Methylphenidate (MPD) is a psychostimulant that is prescribed to treat attention-deficit/hyperactivity
disorder (ADHD) and has been used as a recreational drug. In animal models, repetitive exposure to
methylphenidate can induce a behavioral sensitization. Stimulants are able to change neuronal circuits
in the mesolimbic pathway, and the GABA system is one of the most involved neurotransmitter systems
in this process. Women represent a risk group for psychostimulant abuse because they respond more
strongly, which is probably due to the influence of sex hormones. The objective of the present study was
to investigate the influence of sex hormones on behavioral sentsitization and changes to glutamic acid
decarboxylase (GDA65 and GDA67) isoenzymes and ␣2 GABAA receptor subunit mRNA expression in
the prefrontal cortex and the striatum of rats, as induced by methylphenidate administration (2.5 mg/kg,
i.p.). Female rats were divided into 2 hormonal conditions: ovariectomized and intact group. Repeated
methylphenidate treatment led to behavioral sensitization, which was stronger in females with circulating hormones (intact group). The analysis of mRNA levels in the striatum, in both groups, showed a decline
in GAD65, but not GAD67, transcription after repeated methylphenidate treatment. In the prefrontal
cortex, both GAD65 and GAD67 showed an increase in transcription with repeated methylphenidate
treatment. There was no change in the transcription level of ␣2 GABAA receptor subunits. In conclusion,
it was shown that sex hormones were able to modify behavioral sensitization to methylphenidate and
the drug affected the GABA system in brain areas known to be involved in the development of drug
dependence.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The psychostimulant methylphenidate (MPD) is the most prescribed drug to treat children, adolescents and adults with attention
deficit hyperactivity disorder (ADHD) [1,2], but little is known
about its long-term effects [3]. In recent years MPD has been
more frequently prescribed due to the increasing number of diagnoses. Its widespread clinical use increased the risk of non-medical
use of this drug [4,5]. Because it has pharmacological stimulant
properties similar to amphetamine and cocaine, MPD has the
potential of abuse [6,7]. In fact, these psychostimulants similarly
block dopamine and noradrenaline transporters in pre-synaptic
neurons, thus increasing the availability of these neurotransmitters in the synaptic cleft [8,9]. However, the expression of such
effects depends on a number of variables such as route of administration, prior drug history and diagnostic status [10–13]. Cocaine
∗ Corresponding author at: Pharmacology Division, Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA, Sarmento Leite, 245, 3rd floor – Centro, CEP:
90050-170, Porto Alegre, RS, Brazil. Tel.: +55 51 3003 8821; fax: +55 51 3003 8821.
E-mail address: freese@ufcspa.edu.br (L. Freese).
0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2012.03.017
and amphetamine induce a variety of neuroplastic changes in
brain function and behavior [14,15]. Chronic exposure to low to
moderate doses of MPD causes behavioral sensitization [3,16–18].
Behavioral sensitization is characterized by augmented locomotor
activity to subsequent psychostimulant challenge [19–21]. Sensitization is most commonly manifested as enhanced behavioral
responses and has been associated to drug addiction development
[19]. Behavioral sensitization can be different in males and females
[22,23]. It has already been well established that sex influences
sensitization to psychostimulants in general, and studies with
methylphenidate both in humans and rats also show this trend [24].
Female rats exhibit more robust psychostimulant-induced sensitization [22–24], which has been strongly associated with gonadal
hormones [22,23].
Several neurotransmitter systems are involved in the process
of behavioral sensitization to psychostimulants [7,15]. GABAergic systems can modify the activity of dopaminergic neurons
[14] and besides influencing glutamate and dopamine systems
and indirectly modifying GABA signaling, psychostimulants may
also influence the activity of GABAergic neurons directly. It has
been reviewed that cocaine or amphetamine-treated male animals present variable changes in GAD65 and GAD67 isoenzymes in
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L. Freese et al. / Behavioural Brain Research 231 (2012) 181–186
different brain areas [25]. And it has been demonstrated that
repeated administration of cocaine may induce changes in levels
of enzymes GAD65 and GAD67 in different brain regions, decreases
GADs mRNA ratio in dorsolateral striatum (dSTR) and increases
it in the prefrontal cortex (PFC) of female rats. Additionally, in
this same study it was demonstrated that the GABAergic system
was less impaired by cocaine in intact females in comparison to
ovariectomized rats [25]. GABAA receptors containing ␣2 subunits
that are highly represented in the PFC, which is the main region
involved in signaling motivation, and in the striatum that is related
to motor activation effects of stimulant drugs [26]. These regions
are associated with neuroadaptations, which to mediate facets of
reinforcement [19].
As pointed above, there is pertinent information regarding brain
GABAergic effects by psychostimulants in male animals. There are
not sufficient reports showing the effect of MPD associated to hormonal influence on the sensitization process in female rat and the
participation of the GABA system in response to MPD administration in different hormonal conditions in females needs more
detailing. Thus, the objective of present study was to evaluate both
hormonal influence on the sensitization process and the GABA system involvement in female rats, by comparing the effects of MPD
on GAD65 , GAD67 and ␣2 subunits GABA mRNA expression in the
PFC and dSTR in intact and castrated female rodents.
2. Materials and methods
2.1. Animals
Experiments were conducted with adult female Wistar rats, weighing around
185 g (n = 48), obtained from the animal house of the Universidade Federal de Ciências da Saúde de Porto Alegre, Brazil (UFCSPA). Rats were housed in groups of
five in polypropylene cages (40 × 33 × 17 cm) under standard environmental conditions, such as temperature-controlled rooms (22 ± 2 ◦ C) and a 12-h light/dark cycle
(7:00 am to 7:00 pm). All animals received Nuvilab CR-1 commercial chow phytoestrogens free Nuvital® (Curitiba, Paraná, Brazil) and filtered drinking water ad
libitum. Female rats were randomized to subgroups of ovariectomized (OVX, n = 24)
and intact (INT, n = 24) animals. Bilateral ovariectomy was performed under anesthesia with xylazine (10 mg/kg) and ketamine (75 mg/kg), via i.p., 2 weeks before
starting the experiment. Castration was confirmed by vaginal smears microscopic
analysis. All in vivo experiments followed the guidelines of the International Council for Laboratory Animal Science (ICLAS) and in concordance with the Brazilian law
for the Scientific Use of Animals. This study protocol was approved by the Ethical
Committee for Animal Experimental Procedures of UFCSPA, Porto Alegre, Brazil.
2.2. Drugs
Methylphenidate hydrochloride (Sigma Chemicals, St Louis MO) was diluted in
2% Tween 80 in sterile saline (vehicle) at 2.5 mg/kg/mL. Control rats received saline
solution in the same volume (1 mL/kg). The dosage was selected based on previous
studies [16,27]. This dose produces plasmatic peak in rat similar to those therapeutic
doses used to treat attention deficit hyperactivity disorder (ADHD) in human beings
(∼40 ng/mL) [28]. All drug administrations were performed around 12:00 p.m.
2.3. Procedures
In the first day of the experiment rats in both subgroups were allocated to
receive acute or repeated (5 consecutive days) doses of MPD, 2.5 g/kg, or saline
(CTR), 1 mL/kg, via i.p. After 7 days of washout, rats from acute (ACT) or repeated
(RPT) group received 2.5 g/kg of MPD, i.p. whereas the control group received only
saline (challenge day).
2.4. Behavioral analysis
According to our protocol of sensitization, in the day 1, animals were individually
habituated to the behavioral testing procedure by placement in a photocell cage
(80 × 26 × 22 cm; Alsbarch, Porto Alegre, BR) for 30 min, before the injection of saline
or MPD. After saline or MPD administration, according to the treatment group, rats
were returned to the cages and their horizontal activity was scored for 60 min. The
photocell cage was coupled to a digital counter that was incremented by consecutive
interruption of adjacent beams. In the challenge day, 7 days later, rats from the ACT
e RPT group received both a single dose of MPD, 2.5 mg/kg, i.p., and rats from CTR
group received saline. Immediately after the administration they were tested again
in the photocell cage for locomotor activity behavior during 60 min. At the end of the
behavior test, vaginal smears were collected for estrus cycle phase determination
Fig. 1. Distribution of rats in each estrous cycle stage according to the hormonal
condition and the treatment with methylphenidate (2.5 mg/kg, i.p.). INT, intact; OVX,
ovariectomized; CTR, control; ACT, acute; RPT, repeated.
[29] (see Fig. 1). Afterwards rats were euthanized by decapitation and the striatum
were dissected and stored at −80 ◦ C for further mRNA analysis.
2.5. RT-PCR procedure
Total RNA was extracted from the striatum using TrizolTM Isolation Reagent Kit
(Invitrogen, São Paulo, Brazil) according to the instructions of the manufacturer. The
yield of total RNA was determined by measuring the absorbance (260/280 nm) of
diethylpyrocarbonate (DEPC)-treated water aliquots of samples. All RNAs samples
were resuspended in DEPC-treated water (Invitrogen, São Paulo, Brazil) and stored
at −80 ◦ C. A semi-quantitative RT-PCR technique was used to determine the presence of ␣2 and ␣4 GABAA subunit transcripts in the samples and determine the
levels of GAD65 and GAD67 mRNA. A 3 g aliquot of total RNA extracted from the
hippocampus was reverse transcribed using the SuperScriptTM III Platinum Synthesis System for RT-PCR (Invitrogen Life Technologies) according to the instructions
of the manufacturer.
Relative RT-PCR was performed to measure gene expression of GAD65 and
GAD67 mRNAs and the presence of ␣2 GABAA subunit. Specific oligonucleotides
derived from the coding region of the published sequences of GAD65 (5′ GCTCTACGGAGACTCTGAGAAG 3′ and 5′ CGGTTGGTCTGACAATTCCC 3′ ), GAD67 (5′
TGTGGCGTAGCCCATGGATG 3′ and 5′ ACTGGTGTGGGTGGTGGAAG 3′ ) and ␣2 (5′
GACAATGACCACATTAAGCATCAG 3′ and 5′ TCTTGGCTTCGGCTGGCTTGTTCTC 3′ )
were used. Primer sequences were designed to span intron regions when genomic
sequence data were available, resulting in the predicted 318- and 330-bp sequence,
respectively. The -actin primer set (5′ AGAGGGAAATCGTGCGTGAC 3′ and 5′
CAATAGTGATGACCTGGCCGT 3′ ) that generated a 138-bp product was used as an
internal control. -actin was co-amplified within the same reaction to evaluate
inter-sample variation in cDNA contents and PCR efficiency.
PCR included 2 L of the RT product and was carried out with Taq DNA polymerase (InvitrogenTM Life Technologies Inc.) in a final volume of 25 L, 1.5 mM
MgCl2 , 0.2 M of the specific primer, and 200 M dNTPs. PCR was performed in
a Mastercycler® Personal thermal cycler (Eppendorf, Germany). The amplification
protocol consisted of an initial denaturation step at 94 ◦ C for 3 min, followed by 40
cycles at 94 ◦ C for 1 min, annealing at 60 ◦ C for 1 min, extension at 72 ◦ C for 1 min,
and a final extension at 72 ◦ C for 5 min. The linear amplification range for each
gene was tested on the adjusted cDNA. The less expressed transcripts of GAD65 ,
GAD67 , ␣2 GABAA subunit required >32 PCR cycles for detection. -actin primers
were added when 5 cycles were remaining in the specified gene’s linear amplification range. RT-PCR assays without cDNA samples were carried out as negative
controls. All reactions were performed in duplicate. After amplification, 10 L of the
PCR products was analyzed on a 2.0% ethidium bromide agarose gel. The intensity
of each band was assessed by optical densitometry (ImageMaster® VDS, Amersham
Pharmacia Biotech). An ImageMaster UDS System captured the images® . The optical density (OD) of the GAD65 , GAD67 and ␣2 GABAA subunits and -actin bands
were measured using the program ‘Software Gel Analyzer’. DNA band intensity was
normalized against the corresponding values of the -actin band intensity. Data
are reported as the ratio of the GAD65 /-actin, GAD67 /-actin and ␣2 /-actin these
were used for statistical analysis.
2.6. Data analysis
For analysis of data we used a Two Way ANOVA, considering the factors treatment (CTR, AGD and RPT) and hormonal condition (OVX and INT) for the analysis of
dependent variables: locomotion on the challenge, ratio mRNA of izoenzimas GADs
and ␣2 subunit. The Tukey test was used for post hoc comparisons when appropriate.
L. Freese et al. / Behavioural Brain Research 231 (2012) 181–186
183
RPT) as well as when it was analyzed the hormonal conditions (see
Fig. 4A).
Regarding ␣2 GABAA R subunit mRNA expression in the dSTR,
there was no difference between RPT and ACT, as well as treated
groups and control groups neither related to hormonal condition.
4. Discussion
Fig. 2. Differences in locomotor response as a function of hormones during a 60 min
test, following acute (ACT) or repeated (RPT) treatment with methylphenidate
(2.5 mg/kg, i.p.) in female rats. * P < 0.001 vs. CTR counterparts. # P < 0.001 vs. ACT
counterparts. † P < 0.001 vs. RPT OVX. INT, intact; OVX, ovariectomized.
All data were presented as means ± standard error of the mean. Differences were
considered significant at P < 0.05.
3. Results
3.1. Effects of MPD on locomotor activity
The estrous cycle stage of both intact and ovariectomized female
rats was determined after the final locomotion test, and is shown
in Fig. 1. Some changes in the hormone cycle of ovariectomized
female rats may be due to remnants of ovarian follicles after the
surgical procedure of ovariectomy.
The locomotor activity levels of all groups were recorded during 1 h. MPD elicited a significant (F(2,66) = 109.812; P < 0.001) and
robust increase in the horizontal activity of all three rat subgroups (CTR, ACT and RPT), with the RPT rats exhibiting the
most intense increase in activity also when compared to the
ACT group (P < 0.001). Significant interaction between treatment
with MPD (ACT and RPT) and hormonal condition (OVX and INT)
(F(2,66) = 10.287; P = 0.002) was detected, intact rats had more locomotor behavior due to MPD (P = 0.002) (see Fig. 2).
3.2. Effects of MPD on GADs and on ˛2 GABAA subunit mRNAS
expression in the prefrontal cortex
Both, acute and repeated MDP treatment groups (ACT and RPT)
showed an increase in GAD65 mRNA expression in the PFC when
compared to CTR (see Fig. 3A) (P = 0.005).
As showed in Fig. 3B, GAD67 mRNA levels in the PFC was
increased in RPT group in both (F(2.34) = 5.632, P = 0.008).
There was no difference in PFC ␣2 subunit mRNA expression
in MPD treatment groups. Also, no significant difference between
hormonal condition (INT and OVX) (see Fig. 5).
3.3. Effects of MPD on GADs and ˛2 GABAA subunit mRNAs
expression in the dorsolateral striatum
As shown in Fig. 4A, the RPT group presented a decrease in
GAD65 /ß-actin ratio optical density when compared to ACT group
(F(2.25) = 7.056; P = 0.012) as well as when compared to CTR group
(F(2.25) = 7.056; P = 0.006).
There was no difference in GAD67 mRNA expression between
treated groups and their control counterparts. In the same way,
there was no difference between the treatment types (ACT and
Repeated treatment with MPD produces behavioral sensitization, reproducing previous studies [3,16–18]. In this study, we
showed that MPD may induce behavioral sensitization in female
rats. Also, an interaction between behavioral sensitization to MPD
and the female hormonal status was seen. The effects of behavioral
responses caused by psychostimulants may be directly affected by
sex hormones [30,31]. Preclinical and clinical studies suggest that
females may be particularly vulnerable to the reinforcing effects
of stimulant drugs [32,33]. Female rats show more intense hyperlocomotion behavior and show greater sensitization to cocaine
than males [22]. Here we show that female rats with intact hormonal regulation have increased locomotor activity after repeated
MPD treatment, compared to ovariectomized counterparts. We
also found that the physiological presence of sex hormones may
influence MPD effects, with increased behavioral responses after
a single or repeated MPD administration in non-ovariectomized
(intact) female rats. The increased behavioral locomotor effects
of other psychostimulants, cocaine and amphetamines, has been
described [22,30,34], Understanding the effects of chronic drug use
on GABAergic neurotransmission is critical for understanding the
neuroplasticity associated to psychostimulant effects. GABAergic
neurons play an important role in the modulation of the dopamine
system that is involved in the initiation of behavioral sensitization
[19,21], by reducing dopamine release and inhibiting dopaminergic
activity [35]. The observed upward shifts in the GABA synthesis pathway may be caused by the potentiation of the excitatory
effects of psychostimulants, a consequence of long-term reduction
of the inhibitory capacity of the central nervous system [36] In the
PFC, GABAergic interneurons can regulate dopaminergic and glutamatergic neurotransmission. Animals sensitized to cocaine show
an increase in mPFC GABA release in response to a cocaine challenge
after 1 or 7 days [37].
Since GAD enzyme is expressed only in GABA neurons, it may
be used as a marker for GABAergic function [38]. Changes in GAD65
and GAD67 levels appear to be involved with the processes of
neuroplasticity. The GAD65 isoenzyme plays an important role in
regulating the vesicular pool of GABA and its function is more evident in times of prolonged neuronal activity [45]. GAD65 is located
in the synapses and responds more quickly to demands caused by
GABA neuronal activity. On the other hand, GAD67 has major importance in the production of nonvesicular GABA and it is involved in
the overall metabolic activity the cell [39]. Our findings demonstrate that repeated treatment with MPD significantly increased
the expression of GAD65 and GAD67 in the PFC of female rats,
regardless of the hormonal condition. The expression of GAD65 , the
quick-response enzyme, increased significantly even in the acute
treatment with MPD. These results suggest that there is a process
of neuroplasticity induced by psychostimulants in the GABAergic
system in the PFC, even after a single MPD administration and more
vesicular GABA is recruited under those conditions. Contrary to our
results, the literature points out that at least 4–6 h are necessary to
observe changes in GAD mRNA induced by acute administration of
amphetamine [36] or cocaine [40]. In fact, the effects of MPD are
very different from those of cocaine in intact and gonadectomized
female rats GAD isoenzymes [25]. MPD seems to have a much faster
regulatory control in GAD65 expression.
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L. Freese et al. / Behavioural Brain Research 231 (2012) 181–186
Fig. 3. (A) GAD65 mRNA expression levels in the PFC after acute (ACT) or repeated (RPT) treatment with methylphenidate (2.5 mg/kg i.p.) in female rats. In the lower part
of the figure is shown GAD65 and B-actin mRNA bands expression of from animals groups. (B) GAD67 mRNA expression levels in the PFC after acute (ACT) or repeated (RPT)
treatment with methylphenidate (2.5 mg/kg i.p.) in female rats. Data are reported as means ± SEM. PFC, prefrontal cortex; CTR, control; * P < 0.05 vs. CTR counterparts. In the
lower part of the figure is shown GAD67 and B-actin mRNA bands expression of from animals groups. In the lower part of the figure is shown GAD67 and B-actin mRNA bands
expression of from animals groups. Bands are presented in accordance with the order they appear on the graphic.
Striatal complex is a putative site of action for the motor activating effects of drugs of abuse and associated neuroadaptations and
may mediate several facets of cocaine reinforcement [19]. Repeated
treatment with MPD was also able to induce changes in the striatum of animals. In this study, GAD65 expression mRNA in the dSTR
but not the GAD67 expression mRNA, significantly decreased after
repeated MPD administration compared to controls. The different
effects in the GAD isoenzymes after repeated exposure to MPD
or cocaine in female rats [25], denotes another important diverse
neurochemical effect between psychostimulants.
The ␣2 GABAA R subunit appears to mediate the ability of
dopamine to act as a “gain amplifier” so that, in the absence of
␣2 subunit, facilitation of dopamine transmission by cocaine is no
longer effective in strengthening behavior directed by conditioned
cues [41]. Western blot analysis showed that repeated treatment
with methamphetamine in rats decreased the expression of ␣2
in the nucleus accumbens [42]. However, we show here that the
transcriptional levels of ␣2 subunit of the GABAA receptor in the
striatum and the PFC did not change in female rats. The explanation
for the significant differences of our results with MPD compared
to those who use cocaine or methamphetamine may be due to
different effects of these drugs, leading to different mechanisms
of plasticity. Another explanation may be that the relationship
mRNA/protein is not always linear [43]. Moreover, the studies cited
did not use female rats. The female steroid hormones, like progesterone and estrogen, can influence neurotransmitter systems,
including the GABA system [44].
As GABA is the main inhibitory neurotransmitter in the CNS,
one could expect to correlate the increasing of GABA system activity with lower locomotor activity. Although we cannot confirm
that the increasing of the GAD65 and GAD67 mRNA are closely
related to increasing synthesis or release of GABA we can infer
that if those enzymes increase their expression the system is activated [45]. Interestingly, here we showed that repeated treatment
with MPD increased the expression of GAD65 specifically in the
PFC of female rats, and decreased its expression in the dSTR. As
already known, the PFC is an important area of the limbic circuitry
involved in the control of emotions and cognition and the striatum
Fig. 4. (A) Amount of GAD65 mRNA expression in the dorsolateral striatum (dSTR) after acute (ACT) or repeated (RPT) treatment with methylphenidate (2.5 mg/kg i.p.) in
female rats. In the lower part of the figure is shown GAD65 and B-actin mRNA bands expression of from animals groups. (B) Amount of GAD67 mRNA expression in the
dorsolateral striatum (dSTR) after acute (ACT) or repeated (RPT) treatment with methylphenidate (2.5 mg/kg i.p.) in female rats. Data are reported as means ± SEM. dSTR;
CTR, control. * P < 0.05 vs. CTR counterparts. # P < 0.05 vs. ACT counterparts. In the lower part of the figure is shown GAD67 and B-actin mRNA bands expression of from animals
groups. Bands are presented in accordance with the order they appear on the graphic. In the lower part of the figure is shown GAD67 and B-actin mRNA bands expression of
from animals groups. Bands are presented in accordance with the order they appear on the graphic.
L. Freese et al. / Behavioural Brain Research 231 (2012) 181–186
185
Fig. 5. (A) mRNA expression of ␣2 GABAA subunit in PFC after acute (ACT) or repeated (RPT) treatment with methylphenidate (2.5 mg/kg i.p.) in female rats. In the lower
part of the figure is shown GABAA ␣2 subunit and B-actin mRNA bands expression of from animals groups. (B) mRNA expression of ␣2 GABAA subunit in the dSTR after acute
(ACT) or repeated (RPT) treatment whit methylphenidate (2.5 mg/kg i.p.) in female rats. Data are reported as means ± SEM. dSTR, dorsolateral striatum; CTR, control. In the
lower part of the figure is shown GABAA ␣2 subunit and B-actin mRNA bands expression of from animals groups. Bands are presented in accordance with the order they
appear on the graphic.
is involved in the motor activity [14,19,24,46]. Coincidently here
we showed lower GAD65 expression and higher locomotor activity
in the dSTR, after repeated treatment with MPD. Thus the increasing of the dopaminergic activity and decreasing of GABA activity in
the dSTR could explain in part the stimulatory effect and impulsivity observed after repeated MPD administration. The increasing of
GAD65 and GAD67 mRNA expression after repeated MPD treatment
in the PFC could be related to other behaviors, such as craving.
In summary, this study shows that gonadal hormones play a role
in methylphenidate sensitization in female rats. It also describes
that the decrease in the GAD65 transcript in the striatum, may be
a marker for repeated MPD treatment in animals. Also, it was seen
in the PFC, that the transcription of GAD67 is more selective and
a better marker for repeated MFD treatment. No significant interaction of MPD treatment with the female hormonal condition of
the rats was found, regarding the GAD enzymes expression in the
brain. More studies are necessary to further investigate de the influence of sexual hormones on methylphenidate treatment induced
brain changes. Finally, it is clear that the status of drug of abuse and
the risk of long-term use of MPD will need more investigation, in
respect to its mechanisms of action and that the simple comparison
with the other psychostimulants will not suffice.
Conflict of interest
The authors have no conflict of interest.
Acknowledgments
The authors thank the support from CNPq (HMTB) and the fellowships from CAPES (LF, MFS).
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