Heteromeric nicotinic receptors are involved in the sensitization and
addictive properties of MDMA in mice
Andrés Ciudad-Roberts, Jorge Camarasa, David Pubill* and Elena Escubedo
Department of Pharmacology and Therapeutic Chemistry (Pharmacology Section) and
Institute of Biomedicine (IBUB), University of Barcelona, Barcelona, Spain
Author for correspondence:
Department of Pharmacology and Therapeutic Chemistry,
Faculty of Pharmacy,
University of Barcelona,
Avda. Joan XXIII s/n
Barcelona 08028, Spain.
Short Title: Nicotinic receptors and MDMA rewarding effects.
We have investigated the effect of nicotinic receptor ligands in the behavioral
sensitization (hyperlocomotion) and rewarding properties (conditioned place preference
paradigm, CPP) of 3,4-methylenedioxy-methamphetamine (MDMA) in mice. Each
animal received intraperitoneal pretreatment with either saline, dihydro-β-erythroidine
(DHβE, 1 mg/Kg) or varenicline (VAR, 0.3 mg/Kg), 15 min prior to subcutaneous
saline or MDMA (5 mg/Kg), for 10 consecutive days. On day 1, both DHβE and VAR
inhibited the MDMA-induced hyperlocomotion. After 10 days of treatment, MDMA
induced a hyperlocomotion that was not reduced (rather enhanced) in antagonist-
pretreated animals. This early hyperlocomotion was accompanied by a significant
increase in heteromeric nicotinic receptors in cortex that was not blocked by DHβE or
VAR. Behavioral sensitization to MDMA was highest 2 weeks after the discontinuation
of MDMA treatment. This additional increase in sensitivity was prevented in animals
pretreated with DHβE or VAR. At this time, MDMA-treated mice showed a significant
increase in heteromeric receptors in cortex that was prevented by DHβE and VAR. An
involvement of α7 nicotinic receptors in this effect is ruled out.
MDMA (10 mg/Kg) induced positive CPP that was abolished by DHβE (2 mg/Kg) and
VAR (2 mg/Kg). Moreover, chronic nicotine pretreatment (2 mg/Kg, ip, b.i.d., for 14
days) caused MDMA, administered at a low dose (3 mg/kg), to induce CPP, which
would otherwise not occur. Finally, present results point out that heteromeric nicotinic
receptors are involved in locomotor sensitization and addictive potential induced by
MDMA. Thus, varenicline might be a useful drug to treat both tobacco and MDMA
abuse at once.
Key words: MDMA, nicotinic receptors, sensitization, addiction
MDMA1 is a synthetic drug that has properties of both stimulants and hallucinogens.
Compared to other amphetamine derivatives, MDMA triggers a larger increase in
serotonin and a smaller increase in dopamine release (Johnson et al., 1986). The
behavioral and neurochemical adaptations related to chronic MDMA treatment are
largely unknown. For instance, an increase in the functionality of cortical 5-HT2A and a
decrease in striatal D2 receptors in mice treated with MDMA have been described
(Varela et al., 2011). Many drugs of abuse, at low doses, can increase motor behavior
producing heightened locomotion and exploration (Wise and Bozarth, 1987) and, after
repeated administration, behavioral sensitization can arise from various
neuroadaptations in multiple brain nuclei. This is not only the result of distinct
molecular targets for the drugs, but may also include a differential involvement of
learned associations. It is postulated that the relatively more robust pharmacological
capacity of amphetamine derivatives to release dopamine may induce a form of
sensitization that is more dependent on adaptations in mesoaccumbens dopamine
transmission in comparison to cocaine and morphine sensitization (Vanderschuren and
There is evidence that acetylcholine plays an important role in the hyperlocomotor
activity induced by psychostimulants (Williams and Adinoff, 2008). Dihydro-β-
erythroidine (DHβE), a high-affinity competitive antagonist of α4 subunit-containing
nAChR (nicotinic acetylcholine receptor) inhibits the induction of locomotor
sensitization to d-amphetamine (Karler et al., 1996; Schoffelmeer et al., 2002).
Moreover, knockout mice lacking the β2 nAChR subunit do not self-administer nicotine
(Picciotto et al., 1999) and show less cocaine-conditioned place preference than wild-
type mice (Zachariou et al., 2001). All of these results indicate that heteromeric α4β2
nAChR subtypes appear to play an essential role in nicotine dependence (Govind et al.,
2009); in this regard, an activation of α4β2 nAChR is strongly associated with
dopamine release in the nucleus accumbens (NAcc) (Champtiaux et al., 2003) and with
drug-seeking behavior (Balfour et al., 2000; Picciotto et al., 1999). A particular feature
of nAChR is that chronic exposure to nicotine and other nicotinic ligands induces a
higher level of epibatidine binding (up-regulation) that can lead to an increase in
1 Abbreviations: AUC, area under the curve; CPP, conditioned place preference; DHβE, dihydro-β-erythroidine;
MDMA, 3,4-methylenedioxy-methamphetamine; MLA, methyllycaconitine; NAcc, nucleus accumbens; nAChR,
nicotinic acetylcholine receptors; VAR, varenicline; VTA, ventral tegmental area.
receptor function (functional up-regulation) (reviewed by Gaimarri et al., 2007).
Therefore, the up-regulation of heteromeric nAChR could, via dopamine release,
explain the reinforcing effect of nicotine on the mesolimbic system mediating nicotine
addiction (Balfour et al., 2000).
Studies examining the interactions between nAChR and psychostimulant drugs have
focused primarily on d-amphetamine and cocaine but it is unclear whether such findings
can be extended to other psychostimulants. Previous results from our group (for a
review see Pubill et al., 2011) have demonstrated that nAChR are a pharmacological
target for both methamphetamine and MDMA and are involved in some actions of these
drugs of abuse such as analgesia or locomotor activity (Camarasa et al., 2009), tumor
necrosis factor alpha suppression (Camarasa et al., 2010) and neurotoxicity (Chipana et
al., 2008b; 2008c; Escubedo et al., 2009). We have described the direct and specific
interaction of MDMA with α7 and α4β2 nAChR in mouse brain membranes and
cultured PC12 cells (García-Ratés et al., 2007). The interaction with nAChR occurs at
low micromolar concentrations that can be reached in the mammalian central nervous
system after its administration (Chipana et al., 2008a). Also, similarly to nicotine,
MDMA induces nAChR up-regulation in PC12 cells and in rat brain, where it also
potentiates the regulatory effects of nicotine (García-Ratés et al., 2007; Pubill et al.,
MDMA’s interaction with nAChR might account for some clinical features of this
drug such as fasciculation and muscle cramps, which occur especially in MDMA
abusers after high-dose intake (Klingler et al., 2005). Moreover, tobacco is one of the
most widely consumed drugs and MDMA abusers very often smoke (Scholey et al.,
2004); thus, a pharmacodynamic interaction between nicotine and MDMA can be
expected and could have several consequences that will be suggested at a later point in
This study was undertaken to determine whether nAChR are involved in the
behavioral sensitization and addictive potential of MDMA. DHβE (antagonist) and
varenicline (partial α4β2 nAChR agonist and full α7 nAChR agonist; Mihalak et al.,
2006; Rollema et al., 2007) were associated with MDMA in order to investigate the
involvement of heteromeric nAChRs on its effects. Also, the effect of a chronic
pretreatment with nicotine on MDMA addictive effects was investigated. We focused
on the locomotor hyperactivity induced by MDMA as an indicator of its
psychostimulant effect and on the conditioned place preference (CPP) paradigm to
assess its addictive properties. Also, we investigated the changes in the density of
homomeric and heteromeric nAChRs in determined brain areas as a possible
consequence of the treatment that could be related with the observed behavioural
2. Material and Methods.
2.1. Animals and treatment groups
Data were collected from adult male Swiss CD-1 mice (Charles River, Barcelona,
Spain) weighing 24 to 30 g at the beginning of the experiments (first drug
administration). They were housed three per cage under standard laboratory conditions
(21 ± 1 ºC room temperature and a 12-h light/dark cycle from 8:00 am to 8:00 pm).
Animals had free access to food (standard laboratory diet, PANLAB SL, Barcelona,
Spain) and drinking water. All experimental procedures were conducted between 9:00
am and 5:00 pm and were in compliance with the guidelines of the European
Community Council (86/609/EEC) and approved by the Animal Ethics Committee of
the University of Barcelona under the supervision of the Autonomous Government of
Catalonia. Efforts were made to minimize suffering and reduce the number of animals
In our experiments we administered MDMA at doses closely related to its
recreational use in humans rather than at high doses that would lead to neurotoxic
Mice were assigned randomly to one of six treatment groups: Saline (saline i.p. +
saline s.c.), MDMA (saline i.p. + MDMA s.c.), DHβE (DHβE i.p. + saline s.c.),
DHβE+MDMA (DHβE i.p. + MDMA s.c.), VAR (saline i.p. + varenicline s.c.),
VAR+MDMA (varenicline i.p. + MDMA s.c.). Doses and schedule are detailed below.
Prior to experimentation, all of the animals received two habituation sessions (48 and
24 h before testing) that were intended to reduce the novelty and stress associated with
handling and injection.
Drugs and reagents were obtained from the following sources: 3,4-
methylenedioxymethamphetamine hydrochloride was provided by the National Health
Laboratory (Barcelona, Spain). Varenicline was a gift from Pfizer Laboratories (New
York, USA). Aprotinin, DHβE, methyllycaconitine (MLA), nicotine bitartrate
dihydrate, phenylmethylsulfonyl fluoride and sodium orthovanadate were purchased
from Sigma–Aldrich (St. Louis, MO, USA). [3H]epibatidine was from PerkinElmer
(Boston, MA, USA), while [3H]MLA came from American Radiolabeled Chemicals (St.
Louis, MO, USA). Drugs were dissolved in saline (NaCl 0.9%). All other reagents were
of analytical grade
2.3. Locomotor Activity
This test was used to assess the psychostimulant effects of MDMA along the
treatment and its modulation by nicotinic drugs.
2.3.1. Drug treatment
According to its treatment group allocation, each animal received pretreatment with
either saline (5 ml/Kg), DHβE (1 mg/Kg) or varenicline (0.3 mg/Kg), given
intraperitoneally, 15 min prior to saline or MDMA (5 mg/Kg), given subcutaneously,
for 10 consecutive days. These doses were chosen based on previous reports (Camarasa
et al., 2009; Kim et al., 2011). We administered MDMA at a 5 mg/Kg dose because,
although it is relatively low, it induces robust behavioral activation (Ball et al., 2009).
Once the 10-day repeated treatment phase was completed, all of the animals remained in
their home cages for a 14-day drug-free period (days 11-24). On day 25, all of the mice
were accordingly challenged with either a dose of saline or DHβE or varenicline plus
saline or MDMA to assess for conditioned hyperactivity. Locomotor activity was
measured on days 1, 10 and 25. To evaluate the development of behavioral sensitization
we compared data from day 1 vs day 10 or day 25 of the same group .
On the different testing days and immediately after the i.p. injection (saline or
MDMA), the mice were placed in a plexiglas cage. This cage constituted the activity
box that was placed inside a frame system of two sets of 16 infrared photocells
(LE8811, PANLAB SL, Barcelona, Spain) mounted according to the x, y axis
coordinates and 1.5 cm above the wire mesh floor. The registration of horizontal
locomotor activity then began. Occlusions of the photo beams were recorded and sent to
a computerized system (SedaCom32, PANLAB SL, Barcelona, Spain). The interruption
counts (beam breaks), in a 10-min block, were used as a measure of horizontal
locomotor activity. The locomotor activity of each mouse was monitored over 180 min.
All experiments were conducted between 9:00 am and 3:00 pm. Results are expressed
as cumulative breaks per mouse for 180 min or as AUC (area under the curve), which
was measured as the total changes from baseline at each recording interval over the total
2.4. Radioligand binding experiments
2.4.1. Tissue Sample Preparation
Six hours after the challenge with MDMA on day 10 or on day 25, 5-6 animals per
group were killed by cervical dislocation, then decapitated and the brains rapidly
removed from the skull. Cortex, striata and hippocampus were quickly dissected out,
frozen on dry ice and stored at -80 ºC until use. When required, tissue samples were
thawed and homogenized at 4 ºC in 10 volumes of buffer consisting of 5 mM Tris-HCl,
320 mM sucrose and protease inhibitors (aprotinin 4.5 mg/ml, 0.1 mM PMSF and 1 mM
sodium orthovanadate), pH 7.4, with a Polytron homogenizer. The homogenates were
centrifuged at 15,000 × g for 30 min at 4 °C. The pellets were resuspended in fresh
buffer and incubated at 37 °C for 10 min to remove endogenous neurotransmitters. The
protein samples were subsequently re-centrifuged and washed two additional times. The
final pellets (crude membrane preparations) were resuspended in 50 mM Tris–HCl
buffer plus protease inhibitors and stored at −80 °C until later use in radioligand binding
experiments. Protein content was determined using the Bio-Rad Protein Reagent (Bio-
Rad Labs. Inc., Hercules, CA, USA), according to the manufacturer’s instructions.
2.4.2. [3H]Epibatidine Binding.
[3H]Epibatidine binding was used to label heteromeric nAChR, which in CNS are
mainly α4β2. Binding of [3H]epibatidine to brain membranes from cortex and striatum
was measured as described previously (Chipana et al., 2008b). Briefly, experiments
were carried out in glass tubes containing 1 nM [3H]epibatidine (55.5 Ci/mmol)―at this
concentration primarily α4β2 receptors are labeled (Avila et al., 2003)―and incubation
was carried out for 3 h at 25 °C. The incubation buffer was 50 mM Tris-HCl plus
protease inhibitors. Non-specific binding was determined in the presence of 300 μM
nicotine. Binding was terminated by filtration, and data were treated as described below.
2.4.3. [3H]MLA Binding.
[3H]MLA binding was used to quantify homomeric α7 nAChR. Binding of [3H]MLA
to brain hippocampal membranes was measured as described by Davies et al. (1999).
Briefly, 0.25 ml of membranes (containing 200 μg of brain membranes) was incubated
in borosilicate glass tubes with 2 nM [3H]MLA (60 Ci/mmol), in a final volume of 0.5
ml for 2 h at 4 °C. The incubation buffer consisted of 50 mM Tris–HCl, 120 mM NaCl,
2 mM CaCl2, 1 mM MgSO4 and 0.1% bovine serum albumin. Non-specific binding was
determined from tubes containing 1 μM unlabeled MLA. Incubation was completed by
rapid filtration under vacuum through Whatman GF/B glass fiber filters (Whatman Intl.
Ltd., Maidstone, U.K.) pre-soaked in 0.5% polyethyleneimine. Tubes and filters were
washed rapidly 3 times with 4 ml ice-cold 50 mM Tris–HCl and the radioactivity
trapped was measured by liquid scintillation spectrometry. Specific binding was
calculated as the difference between the radioactivity measured in the absence (total
binding) and in the presence (non-specific binding) of the excess of non-labeled ligand,
and expressed as the percentage of that obtained from saline-treated mice.
2.5. Conditioned Place Preference (CPP) Paradigm.
The place conditioning protocol used was non-biased (Robledo et al., 2004). The
apparatus was composed of three distinct compartments separated by manually operated
doors. The central compartment (corridor) measured 27x10x25 cm (w x d x h) and
served as a thoroughfare between the two pairing sides. The pairing compartments are
20x20x25 cm (w x d x h). One compartment had black and white checkered walls with
a smooth and shiny floor. The other compartment had white and light blue painted walls
and rough flooring. The light intensity within the conditioning chambers was 30 lux.
CPP was performed in three phases: preconditioning, conditioning and test. During the
pre-conditioning phase (day 1), naive or nicotine pre-treated mice were placed in the
middle of the corridor and had free access and roam among the three compartments of
the apparatus for 20 min. The time spent in each compartment was recorded by
computerized monitoring software (Smart Junior, PANLAB SL, Barcelona, Spain).
During the conditioning phase (days 2, 4, 6 and 8), mice were treated with MDMA (3
and 10 mg/kg, s.c.), or saline, 20 min before being confined into one of the two
conditioning compartments for 30 min. On days 3, 5, 7 and 9 of the conditioning phase,
animals received saline and were confined to the opposite compartment. The animals
were exposed to only one pairing per day and treatments were counterbalanced to assure
that some animals received MDMA in the black and white compartment while others
received MDMA in the white and light blue compartment.
Control animals received saline every day. For conditioning studies with DHβE or
varenicline, these drugs or saline were administered intraperitoneally 15 min before
MDMA, at doses previously described as effective in antagonizing nicotine-induced
CPP (2 mg/Kg) (Biala et al., 2010; Walters et al., 2006). The test phase (day 10) was
conducted identically to the preconditioning phase; animals were drug-free and had free
access to the three compartments for 20 min.
To investigate whether nicotine (administered in a previous chronic treatment)
potentiates MDMA-induced CPP, nicotine was given intraperitoneally at a dose of 2
mg/Kg (Dougherty et al., 2008) b.i.d. for 14 days. The day after, nicotine was
withdrawn and preconditioning for CPP was started with MDMA at a dose of 3 mg/Kg
as above. A preference score was expressed in seconds and calculated for each animal
as the difference between the times spent in the drug-paired compartment in the post-
test minus the time spent in the pre-conditioning phase.
2.8. Statistical Analysis
All data are expressed as mean ± standard error of the mean (S.E.M.). Differences
between groups were compared using two-tailed one-way analysis of variance
(ANOVA). Significant (p<0.05) differences were then analyzed by Tukey’s post hoc
test for multiple means comparisons, where appropriate. AUC values were calculated by
nonlinear regression using GraphPAD Prism (GraphPAD software, San Diego, CA,
USA). All statistic calculations were performed using Graph Pad Instat (GraphPad
software, San Diego, CA, USA).
3.1. Effect of nAChR ligands on induction of behavioral sensitization to MDMA
Locomotor activity was used to measure behavioral sensitization to MDMA in the
different treatment groups through time. On day 1 an acute challenge of MDMA (5
mg/Kg) produced significantly greater locomotor activity than saline alone (total breaks
(TB): 3423 ± 267 saline, 4870 ± 244 MDMA, p<0.001). This psychostimulant effect
was fully abolished by pretreatment with DHβE or varenicline (F5,89=6.92, p<0.001, see
figure 1, table 1). DHβE and VAR control groups revealed the absence of effect of these
drugs alone on locomotor activity.
Similarly, on day 10, one-way ANOVA showed a significant effect of treatment
(F5,89=23.04, p<0.001). Daily exposure to MDMA or DHβE+MDMA or
varenicline+MDMA revealed sensitization, expressed as a significant increase in the
psychostimulant effect of MDMA. The inhibitory effect of DHβE and varenicline
observed in the acute challenge of MDMA on day 1 was not present after 10
consecutive days of treatment. Day10/day1 ratio of total breaks (F2,41=175.92, p<0.001;
136.32 ± 3.24% MDMA, 169.23 ± 3.10% DHβE+MDMA and 225.29 ± 2.59%
VAR+MDMA) revealed that these drugs enhanced rather than attenuated this early
sensitization. As on day 1 the animals treated with DHβE/VAR alone denoted the
absence of effect of these antagonists on locomotor activity on day 10.
Behavioral sensitization was monitored up to 2 weeks after the discontinuation of
MDMA treatment. Analysis of results on day 25 to assess conditioned hyperactivity
showed an overall significant difference among treated groups (F5,74=37.25, p<0.001,
see figure 1, table 1). A challenge dose of MDMA induced a stronger behavioral
response than that administered on day 10 (day 25: 8075 ± 404; day 10: 6639 ± 332;
p<0.01). DHβE- or varenicline- pretreated mice showed a response on day 25 that did
not differ from that on day 10 (see figure 1). These results were assessed when
analyzing day25/day10 ratio of total breaks (F2,23=7.12, p<0.01: 118.12±2.49% MDMA,
105.81±3.02% DHβE+MDMA p<0.01 vs MDMA and 108.00±2.86% VAR+MDMA
p<0.05 vs MDMA). Differences between total breaks on day 25 and total breaks on day
10, confirms the results (F2,23=29.15 p<0.001; 1436±163 MDMA, 128±12
3.2. Effect of nAChR ligands on the density of nicotinic receptor subtypes in different
mouse brain areas
Due to the effects observed in locomotor activity experiments, the density of nAChR
was measured in several brain areas of the same animals in order to establish a possible
relationship between such effects and changes in receptor populations. 5 animals of
each treatment group were killed on day 10 after treatment and locomotor activity
measurement, while the rest were kept to obtain the results on day 25.
Treatment with MDMA, DHβE or varenicline for 10 days induced a significant
increase in [3H]epibatidine binding in cortex, compared with those receiving saline
alone (F5,34=2.908, p<0.05) . DHβE also induced such an increase in the striatum. In
this area, MDMA did not modify [3H]epibatidine binding and did not alter the increase
in heteromeric nAChR expression induced by DHβE. Moreover, pretreatment with
varenicline significantly reduced [3H]epibatidine binding in mouse striatum; this was
not altered by MDMA (F5,29=27.231, p<0.001) (Fig. 2B).
After the 14-day drug-free period, the mice treated previously with MDMA (but not
those pretreated only with DHβE or varenicline alone), showed a significant increase in
heteromeric nAChR density in cortex and striatum. The cortical increase in
[3H]epibatidine binding was not present in animals which received pretreatment with
DHβE or varenicline (F3,21=18.936, p<0.001) (Fig. 3A). Only pretreatment with DHβE
prevented the up-regulation induced by MDMA in striatum (F3,23=3.376, p<0.05) (Fig
When analyzing the density of homomeric α7 nAChR in hippocampus, where they
are more highly expressed, no differences in receptor densities, measured as [3H]MLA
binding, were found in MDMA-treated mice (Fig 4).
3.3. Effect of nAChR ligands on the acquisition of MDMA-induced CPP
The CPP paradigm was used to study the effect of the different treatments on the
addictive/rewarding properties of MDMA.
Throughout all experiments, a within-subjects comparison revealed that mice had no
bias. Time (in seconds) spent in both compartments during pre-conditioning were
367.58 ± 56.70 and 326.05 ± 35.69, indicating a lack of preference for either side. This
did not significantly change in the test session (309.12 ± 35.14 and 276.19 ± 28.73)
when saline was paired with both compartments during the conditioning phase.
We first investigated the effect of varenicline and DHβE in the CPP induced by
MDMA (10 mg/kg). On the test day (day 10, post-conditioning), one-way ANOVA
revealed a significant effect of treatment (F5,36 = 4.56, p<0.01). The ability of MDMA to
produce a CPP was assessed while some mice were under the influence of DHβE or
varenicline (2 mg/kg) treatment, administered 15 min before the MDMA dose. Both
reduced MDMA's ability to produce a CPP, fully blocking MDMA's effects (p<0.05 for
varenicline and p<0.01 for DHβE vs. MDMA-treated mice) (Fig 5B). Neither DHβE
nor varenicline alone had any effect on CPP.
During the pre-conditioning phase and test day we measured the distance and speed
of travel in each of the two compartments. Results corresponding to the drug-paired
compartment are shown in Table 2 and demonstrate that treatment with MDMA during
the conditioning phase induces an increase in locomotor activity in the test day that is
not present in animals pretreated with varenicline or DHβE. This increase in locomotor
activity was not accompanied by an increase in speed and confirms a psychostimulant
effect in these animals.
To explore the effect of a chronic nicotine treatment on the addictive behavior caused
by a low dose of MDMA (3 mg/Kg) which is not supposed to induce CPP when given
alone (Robledo et al., 2004), we pretreated mice with nicotine at a dose of 2 mg/Kg,
given subcutaneously (b.i.d.) for 14 days. This treatment induced a significant increase
in α4β2 nAChR density in the striatum (147.98 ± 13.13%, nicotine-treated vs 100.00 ±
10.56%, saline-treated, p<0.05, Student’s t test). This nicotine treatment schedule did
not induce a significant CPP on its own (Dougherty et al., 2008) and, therefore, at the
end of the nicotine treatment, animals did not show preference for either of the two
compartments (445.85 ± 69.28 vs 551.02 ± 27.82). Repeated nicotine administration
during the 14 days prior to pre-conditioning led to a decreased MDMA threshold for
CPP. As reflected in Fig. 6, when animals were exposed to chronic nicotine
pretreatment, they showed a positive preference score at a dose of MDMA (3 mg/Kg)
that proved to be ineffective when administered alone (F2,23 = 5.808, p<0.01).
This study examines the involvement of heteromeric nAChR in the behavioral
sensitization as well as the addictive potential of MDMA in mice. The results indicate
that an antagonism or a partial agonism on nAChR reduces the addiction, blocks the
acute locomotor effects and changes the development of sensitization induced by
MDMA. α4β2 nAChR appear to mediate these effects given that DHβE and varenicline,
but not MLA (data not shown), antagonized the acute effects of MDMA. In fact,
previous studies (Walters et al., 2006) have demonstrated that MLA at doses of 5 and 10
mg/Kg (s.c), does not inhibit nicotine-induced CPP, ruling out an involvement of the α7
nAChR in this behavior.
The psychomotor stimulant effect of MDMA is considered subsequent to an
extracellular increase in DA and 5-HT in the NAcc and VTA (Bankson and
Cunningham, 2001). In a previous study we demonstrated the involvement of nicotinic
receptor subtypes in the hyperlocomotion induced by methamphetamine (Camarasa et
al., 2009). Here we report that the stimulant effects of an acute dose of MDMA are
blocked by antagonists acting on α4β2 nAChR. Nicotinic agonists can differentially
affect neurotransmitter release in a given brain region and the magnitude of such
responses will largely be determined by the subtype selectivity of the agonist (Rao et al.,
2003). Nicotine activates nAChR localized in the dopaminergic nerve terminals in the
nucleus accumbens and elicits a complex pattern of inhibitory–stimulatory effects on
locomotion (Avale et al., 2008).
Although there are subtle differences between MDMA and other commonly abused
amphetamines, a clear overlap in the behavioral pharmacology of MDMA and other
amphetamine-like compounds can be found, especially in the induction of behavioral
excitation. In rodents, this effect, called behavioral sensitization, persists many months
after the last administration, thus mimicking long-term sensitivity to drugs observed in
human addicts. Expression of this persistent drug-induced behavioral sensitization has
been suggested to contribute to craving and high relapse rates in addicts (Robinson and
Berridge, 2003). Studies of the neurobiological basis of behavioral sensitization have
focused on the increased capacity of these drugs to release dopamine in the midbrain
dopamine system (Cadoni et al., 2000) although multiple limbic-associated areas such
as the prefrontal cortex provide the excitatory cortical innervation to the NAcc (Kita and
Kitai, 1990). This dopaminergic system mediates locomotor stimulation as well as the
ability of drugs to elicit craving and lead to abuse.
When MDMA was administered daily for 10 consecutive days, there was an increase
in the hyperlocomotion induced by this drug on day 10 respect with that measured on
day 1 (early behavioral sensitization). These results are in agreement with those
previously described in rats (Kalivas et al., 1998) demonstrating that repeated
administration of MDMA over the course of ten days produces sensitization to the
behavioral stimulant effects of MDMA. Furthermore, the behavioral sensitization in
mice was found to be highest after a 2 week-period following the discontinuation of
MDMA treatment, (a challenge dose of MDMA showed a stronger behavioral response
than on day 10) demonstrating that the treatment schedule of MDMA used in this study
induces not only an early but also a delayed sensitization that can be modulated by
drugs acting on α4β2 nAChR.
Neither DHβE nor varenicline blocked but rather enhanced the development of early
behavioral sensitization by MDMA, conversely to the inhibitory effect observed in the
acute challenge (day 1). When comparing the ratios D10/D1 of the different groups, a
potentiation was revealed for those treated with MDMA plus DHβE or varenicline. In
other words, the groups receiving MDMA plus the nicotinic ligand showed a day-to-day
greater increase in locomotion than the group receiving MDMA alone.
The increased delayed sensitization to MDMA was prevented when it was
administered together with either the α4β2 nAChR antagonist (DHβE) or the partial
agonist (varenicline). It is known that nAChR ligands regulate sensitization to stimulant
drugs such as d-amphetamine and cocaine. For instance, DHβE, a high-affinity
competitive antagonist of α4 subunit-containing nAChR, attenuates the induction of
locomotor sensitization to d-amphetamine, cocaine, ephedrine and methylphenidate in
mice and rats (Karler et al. 1996; Miller and Segert 2005; Schoffelmeer et al. 2002;
Woorters and Bardo, 2009). Additionally, the sensitizing effect of acute nicotine on
amphetamine-stimulated behavior and dopamine efflux requires activation of β2
subunit-containing nAChRs (Kim et al., 2011).
Varenicline is an effective aid in smoking cessation. This drug, by acting on α4β2
nAChR, stimulates dopamine release when the basal tone is depressed and
simultaneously blocks the effects of a full agonist when simultaneously present. Partial
agonists aim to provide a low-to-moderate level of dopamine stimulation to reduce
craving and withdrawal symptoms. When varenicline is administered to nicotine-
sensitized rats, it reduces the expression of nicotine sensitization (Zaniewska et al.,
2008). Similarly, in our experiments, varenicline inhibited the increase in the delayed
sensitization observed on day 25.
Due to the described dynamic plasticity of nAChR after treatment with nicotinic
ligands, we assessed the density of heteromeric (mainly α4β2) and homomeric α7
receptors through radioligand binding studies. The results showed that early
sensitization on day 10 was accompanied by changes in α4β2 nAChR density in certain
brain areas. MDMA induced in cortex, but not in the striatum, a significant increase in
α4β2 nAChR that was not blocked by DHβE or varenicline. However, the results on day
25 correlate with the in vivo effects: although these animals had a 14-day drug-free
period, the increased α4β2 nAChR density in cortex and striatum was still present in the
MDMA group, but not in the animals co-treated with DHβE. Varenicline appears to do
the same in the cortex. From these results it can be deduced that the α4β2 nAChR
subtype is involved in the early and delayed sensitization elicited by MDMA. If
treatment leads to an increase in α4β2 nAChR subtype population in cortex, the
sensitization takes place. By contrast, when this up-regulation is prevented, sensitization
is attenuated. The role of the cortex in sensitization is not an exception as it is known
that the prefrontal cortex and the hippocampus exhibit converging projections to the
NAcc and have functional reciprocal connections via indirect pathways (Day et al.,
1991; Goto and Grace, 2008). Medial prefrontal neurons, including those projecting to
the NAcc (McGinty and Grace, 2008), are also excited by conditioned stimuli
(Laviolette et al., 2007) and Ball et al (2009) demonstrating that long-lasting locomotor
sensitization to MDMA is accompanied by reorganization of synaptic connectivity, not
only in NAcc, but also in the medial prefrontal cortex.
Effects derived from changes in α7 nAChR population can be ruled out from present
binding studies. The difference between the effects of DHβE and varenicline can be
explained by their different pharmacological profile.
Once the correlation between nAChR and behavioral sensitization to MDMA was
demonstrated, we examined the effect of α4β2 nAChR ligands as well as that of a
nicotine chronic treatment on the CPP score induced by MDMA. In this study we
provide evidence that MDMA at a dose of 10 mg/kg, but not 3 mg/Kg, causes positive
CPP in mice. These results are in agreement with those of Salzmann et al. (2003) and
Robledo et al. (2004). Bilsky et al. (1998) demonstrated that the CPP induced by
MDMA was effectively blocked by the dopamine release inhibitor CGS10746B. These
results and those of Vidal-Infer et al. (2012) demonstrate that, in mice, the dopaminergic
system is involved in the acquisition and expression of MDMA-produced CPP.
Moreover, results of the present study provide pharmacological evidence of the
involvement of the α4-containing nAChR in the CPP induced by MDMA, as this effect
was antagonized by DHβE and varenicline.
Acute nicotine challenge induces behavioral sensitization to amphetamines (Birrell
and Balfour, 1998, Jutkiewicz et al., 2008) and consequently can enhance its addictive
potential. In this study we used a chronic nicotine treatment in order to increase the
density of α4β2 nAChR (Dougherty et al., 2008). It is important to note that nicotine
treatment took place previously and this drug was not present during the CPP
experiments with MDMA, avoiding any interaction on the test day. Abstinence signs of
nicotine are dose-dependent and appear at doses equal to or higher than 6.3–8
mg/kg/day (Isola et al., 1999, Gould et al., 2012) and not at 6 mg/kg/day or lower
(Damaj et al., 2003), as in our experiments. These signs last for a maximum of 3-4 days
(Zhang et al., 2012) and are supplemented with deficits in contextual learning (Gould et
al., 2012). In the present study, sustained exposure to nicotine significantly increased
MDMA rewarding in the CPP paradigm. While MDMA at a low dose (3mg/kg) did not
induce CPP on its own, this dose of MDMA showed a very significant preference score
in nicotine-pretreated mice.
As in the behavioral sensitization experiments, this increase in the CPP score caused
by MDMA runs parallel to an increase in α4β2 nAChR density induced by nicotine,
pointing to an up-regulation of these receptors as an additional factor in MDMA’s
reinforcing effect. The up-regulated nAChR could mediate enhanced synaptic
transmission when stimulated by local and brief releases of ACh at synapses.
Stimulation of dopamine neurons in the VTA via the α4β2 nAChR leads to an increase
of dopamine in the NAcc that plays a crucial role in drug reward as measured by CPP
(Di Chiara and Imperato, 1988). Consequently, the modulation of dopamine release by
means of α4β2 nAChR activation could result in a modification of the CPP induced by
MDMA. Although animals were not under the effect of nicotine when tested in the CPP
paradigm, and despite the very low dosage of this stimulant administered during the
pretreatment phase, we cannot rule out an influence of nicotine withdrawal in the first
days of the conditioning phase.
The influence of chronic nicotine treatment on MDMA effects extends not only to
CPP but also to its hyperlocomotion properties. In previous studies (Camarasa et al.,
2009) we have described that nicotine, when administered in a chronic low-dose
schedule, significantly potentiates the methamphetamine-induced increase in locomotor
activity and rearing. These results suggest that up-regulation of nAChR leads to a very
significant potentiation of the increase in locomotor activity induced by this drug.
Similar results were obtained for MDMA-induced hyperlocomotion using the same
nicotine pretreatment than in the study with methamphetamine (a 30% potentiation,,
A great number of MDMA consumers also smoke concomitantly (Scholey et al.,
2004). In view of results obtained in the present paper it can be deduced that smoking
can increase neuronal sensitization to MDMA and its addictive potential, making
MDMA-users more susceptible to addiction. Although further research must be done on
this subject, our results suggest that α4β2 nAChRs are a potential target towards treating
nicotine and MDMA polyabuse. Although DHβE is a useful pharmacological tool for
preclinical studies on nAChR, it is not adequate for clinical use due to its toxicity: it can
produce neuromuscular blockade, hypotension and has a vey narrow dosage window
(the i.p. DL50 in mice is 4.5 mg/kg, Megirian et al., 1955). Also DHβE, as a pure
antagonist, can precipitate nicotine abstinence syndrome (Malin et al., 1998).
Conversely varenicline, as a marketed drug for smoking cessation with a good security
profile, should be taken into consideration as a possible candidate drug.
In summary, although it is well known that nAChRs are a pharmacological target for
understanding the neurotoxic effects of amphetamine derivatives (Chipana et al.,
2008c), they are also involved in other behavioral effects of these drugs such as
hyperlocomotion and addictive properties. This paper demonstrates the involvement of
specific α4-containing nAChR subtypes by using specific modulators of these receptors.
Our results point out that effects induced by MDMA such as locomotor sensitization
and addictive potential, both related with the release of dopamine, are modulated by
DHβE and varenicline. Consequently, varenicline, a commercial drug used to treat
tobacco addiction, could also be considered for treating MDMA abuse. Finally, these
results may have clinical implications because MDMA abusers are often smokers; in
this regard, varenicline would be the first useful drug to simultaneously treat both
tobacco and MDMA abuse.
All authors disclose any actual or potential conflict of interest including financial,
personal or other relationships with other people or organizations that could
inappropriately influence the present work. All authors reviewed the content and
approved the final version.
JC and EE were responsible for the study concept and design. AC, JC and DP
contributed to the acquisition of animal data. JC and DP performed data analysis. EE
interpreted findings and provided critical revision of the manuscript.
The authors wish to acknowledge A. Ciudad-Roberts for language revisions on the
manuscript. This study was supported by the following grants from: the regional
authorities Generalitat de Catalunya (2009SGR 977) to DP; the Spanish drug initiative
Plan Nacional sobre Drogas (2008/003) to DP and (2010/005) to JC; and the Spanish
Ministerio de Ciencia e Innovación (SAF2010-15948) to EE. The funding sources had
no involvement in writing, providing advice on or submitting this report.
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Cumulative breaks after 180 min for the effect of saline, DHβE (1 mg/Kg), or
varenicline (VAR) (0.3 mg/Kg) on saline/MDMA (5 mg/Kg)-induced hyperlocomotion.
Locomotor activity was measured on day 1 (acute challenge), day 10 (after a daily dose
for ten days) and day 25 (acute challenge of saline, DHβE or varenicline plus saline or
MDMA after 14-day withdrawal). Data are expressed as mean ± SEM. *p<0.05,
**p<0.01, ***p<0.001, significantly different from day 1 of the same treated group.
##p<0.01 significantly different from day 10 of the same treated group. φφφ p<0.001
significantly different from saline day 1.
Effect of treatment with saline, DHβE (1 mg/Kg), or varenicline (VAR) (0.3 mg/Kg)
plus saline or MDMA (5 mg/Kg) during 10 consecutive days on α4β2 nAChR density
(measured as [3H]epibatidine binding) in mouse cortex (panel A) or striatum (panel B).
Data are expressed as mean ± SEM from the values obtained from 5-6 animals per
group. *p<0.05 and **p<0.01, significantly different from saline-treated group.
Effect of a 14 day withdrawal after a 10 consecutive day treatment with saline, DHβE (1
mg/Kg), or varenicline (VAR) (0.3 mg/Kg) plus saline or MDMA (5 mg/Kg) on α4β2
nAChR density (measured as [3H]epibatidine binding) in mouse cortex (panel A) or
striatum (panel B). On day 25, mice were killed 6 h after receiving the assigned
treatment and their brains were used for this experiment. Data are expressed as mean ±
SEM from the values obtained from 5-6 animals per group. *p<0.05 significantly
different from saline-treated group.
Effect of MDMA (5 mg/Kg) alone for 10 consecutive days (day 10) or after a 14 day
withdrawal period (day 25) on α7 nAChR density (measured as [3H]MLA binding) in
mouse hippocampus. Data are expressed as mean ± SEM from the values obtained from
5-6 animals per group
Effect of DHβE (2 mg/Kg) and varenicline (VAR) (2 mg/Kg) alone and on MDMA (10
mg/Kg)-induced conditioned place preference. The x-axis represents the treatment
group and the y-axis represents the preference score (test day minus preconditioning
day) in seconds. **p<0.01, significantly different from saline-treated group; #p<0.05
and ##p<0.01, significantly different from the corresponding value of MDMA-treated
Effect of a 14 day chronic nicotine pretreatment (2 mg/Kg, b.i.d.) on the conditioned
place preference assay on MDMA (3 mg/Kg). Data are expressed as mean ± SEM.
**p<0.01, significantly different from saline- or MDMA-treated groups.