ORIGINAL INVESTIGATION

MDMA administration during adolescence exacerbatesMPTP-induced cognitive impairment and neuroinflammationin the hippocampus and prefrontal cortex

Giulia Costa & Nicola Simola & Micaela Morelli

Received: 13 January 2014 /Accepted: 7 March 2014# Springer-Verlag Berlin Heidelberg 2014

AbstractRationale We have recently shown that chronic exposure to3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”)of adolescent mice exacerbates dopamine neurotoxicity andneuroinflammatory effects elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the substantia nigraand striatum at adulthood.Objectives The present study investigated whether the ampli-fication of MPTP effects by previous treatment with MDMAextends to the limbic and cortical regions and consequentlyaffects cognitive performance.Methods Mice receivedMDMA (10mg/kg, twice a day/twicea week) for 9 weeks, followed by MPTP (20 mg/kg×4 ad-ministrations), starting 2 weeks after MDMA discontinuation.Complement type 3 receptor (CD11b) and glial fibrillaryacidic protein (GFAP) were evaluated by immunohistochem-istry in both the hippocampus and the medial prefrontal cortex(mPFC) to measure microglia and astroglia activation. Theseneurochemical evaluations were paired with an assessment ofcognitive performance by means of the novel object recogni-tion (NOR) and spontaneous alternation tasks.

Results MPTP administration to MDMA-pretreated miceelicited a stronger activation of CD11b and GFAP in boththe hippocampus and the mPFC compared with either sub-stance administered alone. Furthermore, NOR performancewas lower in MDMA-pretreated mice administered MPTPcompared with mice that received either substance alone.Conclusions These results demonstrate that MDMA–MPTPnegative interactions extend to the limbic and cortical regionsand may result in cognitive impairment, providing furtherevidence that exposure to MDMA may amplify the effectsof later neurotoxic insults.

Keywords Amphetamines . Astroglia . CD11b . Dopamine .

Drug of abuse . Ecstasy . GFAP .Microglia . Novel objectrecognition task . Spontaneous alternation

Introduction

3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”)is an amphetamine-related drug that may elicit significantneurobehavioral adverse effects, some of which have beenhypothes ized to depend on i t s neuro toxic andneuroinflammatory potential (Barrett et al. 2006; Cadet et al.2007; Capela et al. 2009; Parrott 2002).

Besides showing the detrimental effects of MDMA alone,studies in experimental animals have demonstrated that thisdrug may amplify the neurotoxicity and neuroinflammationelicited by other psychoactive/toxic substances. Such interac-tion has been reported, for instance, between MDMA andethanol, as well as between MDMA and caffeine(Hernandez-Rabaza et al. 2010; Izco et al. 2007; Khairnaret al. 2010; Ros-Simò et al. 2012, 2013). The study of howMDMA modulates the toxic effects of other substances ap-pears to be a “hot topic” in neuropharmacology andneurotoxicology for two major reasons. First, MDMA is

G. Costa :N. Simola :M. Morelli (*)Department of Biomedical Sciences, Section ofNeuropsychopharmacology, University of Cagliari, Via Ospedale 72,09124 Cagliari, Italye-mail: morelli@unica.it

M. MorelliCentre of Excellence for Neurobiology of Dependence, University ofCagliari, Cagliari, Italy

M. MorelliNational Research Council (CNR) Neuroscience Institute, Cagliari,Italy

M. MorelliNational Institute of Neuroscience (INN), University of Cagliari,Cagliari, Italy

PsychopharmacologyDOI 10.1007/s00213-014-3536-z

largely consumed by adolescent people, and adolescence is acritical period for the development of the brain, which un-dergoes major developmental changes, particularly at the levelof the prefrontal cortex (PFC) and limbic regions (Casey et al.2000; Piper and Meyer 2004). In fact, the adolescent brainappears to be particularly vulnerable to the long-term noxiouseffects of exogenous substances, including drugs of abusesuch as MDMA (Cadoni et al. 2013; Daza-Losada et al. 2009;Sisk and Zehr 2005; Spear 2000). Second, a number ofclinical reports have shown a significantly higher incidenceof neurological conditions causing cognitive impairment andneurodegenerative disorders, such as Parkinson’s disease (PD),in people who have abused amphetamine-related drugs(Callaghan et al. 2010; Christine et al. 2010; Garwood et al.2006; Parrott et al. 2003, 2004). In this regard, it is worthmentioning that we have recently demonstrated an amplifica-tion of the neuroinflammatory and neurotoxic effects elicitedby 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), atoxin known to induce PD, in the substantia nigra parscompacta (SNc) and striatum of adult mice treated withMDMA during adolescence (Costa et al. 2013). These resultsprovide preclinical evidence that early exposure to MDMAmay render the dopaminergic system more vulnerable to laterneurotoxic insults. Thus, preclinical studies in the rat haveshown how MDMA is able to produce neuroinflammatoryresponses, characterized by microglial activation and by anincrease in both interleukin-1β (IL-1β) and its precursor pro-tein (pro-IL-1β) in the frontal cortex (Orio et al. 2004), and byan increase in astroglial levels in the hippocampus (López-Rodríguez et al. 2013). In this regard, it is also worth men-tioning that studies in experimental models of PD have sug-gested that neuroinflammatory mechanismsmay participate inthe cascade of events leading to dopaminergic cell death(Barcia et al. 2003; Hirsch and Hunot 2009), which wouldprovide further support to the hypothesis of a link betweenMDMA consumption and later PD. Moreover, MDMA pro-motes the synthesis of free radicals and reactive oxygen spe-cies (ROS) (Barbosa et al. 2012; Hondebrink et al. 2011),wh i ch may con t r i bu t e to t he exace rba t i on o fneuroinflammatory responses elicited by other substances thatare toxic to dopaminergic neurons, such as MPTP.

The possibility that MDMA might induce neurotoxicity issupported by findings obtained in humans that employedfunctional magnetic resonance imaging (fMRI) and positronemission tomography (PET). These studies have shown thatMDMA may affect the metabolism of brain areas involved inthe regulation of cognition, mood, and memory, such as thehippocampus and the PFC (Becker et al. 2013; Bolla et al.1998; Bosch et al. 2013; Daumann et al. 2004, 2005; Moelleret al. 2004; Parrott and Lasky 1998). Moreover, some studieshave shown the existence of cognitive deficits in MDMAusers who began taking the drug during adolescence(Jacobsen et al. 2004; Weinborn et al. 2011). However, no

exhaustive preclinical evidence is currently available on theexistence of a relationship between MDMA-induced neuroin-flammation in brain areas involved in the regulation of cog-nition and cognitive decline.

Based on these considerations, and on our previous inves-tigation of MDMA–MPTP interactions (Costa et al. 2013),this study was undertaken to elucidate whether MDMA mayamplify the neuroinflammatory effects elicited by MPTP inthe hippocampus and medial PFC (mPFC), and if thisneuroinflammatory response may be associated with cogni-tive deficits. The hippocampus and mPFC were selected be-cause both areas are involved in the regulation of cognition, asshown by preclinical and clinical evidence (Aggleton andBrown 1999; Brewer et al. 1998; Eldrige et al. 2000). More-over, studies in experimental animals have demonstrated thatboth MDMA and MPTP elicit neuroinflammatory responsesin the hippocampus and mPFC (Busceti et al. 2008; Kokovayand Cunningham 2005; Straiko et al. 2007; Sy et al. 2010;Wang et al. 2009), and this suggests that a noxious interactionMDMA and MPTP might take place in these brain areas.Finally, these areas were chosen in light of the above-mentioned human studies showing alterations in the hippo-campal and cortical metabolism of MDMA consumersdisplaying cognitive deficits (Becker et al. 2013; Bolla et al.1998; Bosch et al. 2013; Daumann et al. 2004, 2005; Jacobsenet al. 2004; Moeller et al. 2004; Parrott and Lasky 1998;Weinborn et al. 2011). Neuroinflammation was assessed bymeasuring microglial and astroglial activation through immu-noreactivity for complement type 3 receptor (CD11b) andglial fibrillary acidic protein (GFAP), respectively. Moreover,in an attempt to link neurochemical changes with behavioralmodifications, this study investigated whether cognitive im-pairment occurred in mice treated with MDMA and MPTP,alone or combined, by means of two memory tasks, the novelobject recognition (NOR) and the spontaneous alternationbehavior in a Y-maze, which were both performed throughoutthe MDMA treatment and after MPTP administration.

Materials and methods

Animals

Male C57BL/6J mice (Charles River, Calco, Italy) weighing20–23 g at the beginning of the experiments were used. Micewere maintained at constant temperature (21±1 °C) under a12-h light/dark cycle with food and water available ad libitum.Experimental procedures were approved by the ethical com-mittee of the University of Cagliari, in compliance with Italianguidelines for care and use of experimental animals (DL 116/92) and the European Communities Council Directive(2010/63/EEC). Efforts were made to minimize the numberof animals used and maximize humane treatment.

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Drugs

MDMAwas synthesized at the Department of Environmentaland Life Sciences of the University of Cagliari, as reportedelsewhere (Frau et al. 2013).MPTPwas purchased from SantaCruz Biotechnology, CA, USA. MDMA was dissolved insaline, whereas MPTP was dissolved in distilled water. Bothdrugs were administered intraperitoneally (i.p.) in a volume of10 ml/kg.

Treatment

Late adolescent mice (8 weeks old) were repeatedly treatedwith either MDMA (10 mg/kg) or vehicle according to a 9-week administration schedule, as described elsewhere (Costaet al. 2013) (Fig. 1). Repeated treatment took place frompostnatal day (PND) 56 to PND 119. In detail, each mousereceived two administrations a day, separated by an intervalranging between 4 and 6 h, twice a week, on the second andfifth day of each week, for 9 weeks, and a total of 36 admin-istrations of either MDMA or vehicle. Experimental groupswere composed as follows: MDMA-treated mice, n=18–19;vehicle-treated mice, n=17–18. During MDMA treatment,performance in the NOR test was evaluated at weeks 1, 4,and 9 and then reassessed 2 weeks after MDMA discontinu-ation. NOR performance was always measured on the sixthday of the week. The same mice were also evaluated forspontaneous alternation behavior in a Y-maze, 24 h after the

NOR task (on the seventh day of the week) at the same timepoints indicated above. Two weeks after MDMA discontinu-ation, from PND 133 to PND 137, mice (19 weeks old) weredivided into four groups and challenged with either MPTP(20 mg/kg) or vehicle, once a day for 4 consecutive days.Experimental groups were composed as follows: vehicle+vehicle, vehicle+MPTP, MDMA+vehicle, MDMA+MPTP(n=5–10). Mice were evaluated again for their NOR perfor-mance and spontaneous alternation in a Y-maze at 24 or 48 h,respectively, after the last MPTP or vehicle injection. The dayafter the completion of behavioral experiments, mice wereanesthetized with chloral hydrate, transcardially perfused with4 % paraformaldehyde in phosphate buffer (0.1 M, pH 7.4),and their brains were removed and processed for immunohis-tochemistry (Costa et al. 2013).

Immunohistochemistry

For each mouse, three sections from the hippocampus (ante-rior–posterior, 1.46–1.82mm from bregma) and three sectionsfrom the mPFC (anterior–posterior, 2.22–1.78 mm from breg-ma), according to the atlas of Paxinos and Franklin (2008),were used for each analysis.

Coronal brain sections (50-μm thick) were cut on avibratome. Free-floating sections of hippocampus and mPFCwere incubated overnight with CD11b and GFAP antibodies(monoclonal rat anti-CD11b, 1:1,000, Serotec, Oxford, UK;monoclonal mouse anti-GFAP, 1:400 Sigma, Milan, Italy) to

Fig. 1 Experimental design. Late adolescent mice were repeatedly treat-ed with either MDMA (10 mg/kg i.p.) or vehicle (i.p.), according to a 9-week administration schedule. Repeated treatment took place from PND56 to PND 119. In detail, each mouse received two administrations a day,separated by an interval ranging between 4 and 6 h, twice a week, for9 weeks. The performance of mice in the NOR task and spontaneousalternation behavior was evaluated during chronic MDMA treatment andafter its discontinuation. Two weeks after MDMA discontinuation, mice

(19 weeks old) were challenged with either MPTP (20 mg/kg, i.p.) orvehicle (i.p.), once a day for 4 consecutive days, from PND 133 to PND137. Mice were tested again for NOR performance and spontaneousalternation behavior, sacrificed the day after the completion of behavioralexperiments, and their brains processed for immunohistochemistry. Thenumbered bars indicate the day of the week when the mice received thedrug (ticked bar) or performed the cognitive tasks (dashed bar). MDMA-treated mice, n=18–19; vehicle-treated mice n=17–18

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evaluate neuroinflammatory responses. After incubation withprimary antibodies, slices were incubated in proper biotinyl-ated secondary antibodies (Vector, Peterborough, UK), andthe avidin–peroxidase protocol (ABC, Vector, Peterborough,UK) was applied for visualization, using 3,3 -diaminobenzi-dine (Sigma,Milan, Italy) as a chromogen. Sections were thenmounted onto gelatin-coated slides for microscopicevaluation.

Analysis of CD11b immunoreactivity

The cornu ammonis (CA) regions of the hippocampus (CA1,CA2, and CA3) and the whole prelimbic/infralimbic areas ofthe mPFC were analyzed for both hemispheres. Images weredigitized with a video camera (Pixelink® PL-A686, Ottawa,Canada), captured at ×20 real magnification in gray scale, andthen evaluated with the Scion Image analysis software (Scion,Frederick, MD, USA). A threshold, set above the mean value±S.E.M. of the background, was applied for backgroundcorrection. The area occupied by values above the thresholdwas automatically calculated inside each frame. No significantdifferences in CD11b levels were obtained between the threeregions of the hippocampus; therefore, the data obtained werepooled together. For each level of either the hippocampus orthe mPFC, the obtained value was first normalized with respectto vehicle, and values from different levels were averagedthereafter.

Analysis of GFAP immunoreactivity

GFAP immunoreactivity was performed in the CA1, CA2,and CA3 regions of the hippocampus from both hemispheresand in the whole left and right prelimbic/infralimbic areas ofthe mPFC. Images were captured and evaluated with thePixelink® software. The absolute number of GFAP-positivecells was obtained separately for each level of the hippocam-pus and mPFC, and no significant differences in the number ofGFAP-positive cells were observed among the three regionsof the hippocampus; therefore, the data obtained were pooledtogether. For each level of either the hippocampus or themPFC, the obtained value was first normalized with respectto vehicle, and values from different levels were averagedthereafter.

Behavioral tests

Novel object recognition (NOR) task

Evaluation of NOR performance is widely used for assessingnonspatial working memory in rodents (Ennaceur andDelacour 1988). NOR experiments were performed in a Plex-iglas cage (length 25.5 cm, width 19 cm, height 14 cm) with asawdust-covered floor. Objects to be discriminated were made

of plastic and differed as to their shape and color. Moreover,objects had no genuine significance and had not been previ-ously associatedwith either rewarding or aversive stimuli. Theexperimental procedure consisted of three phases: habituation(S0), acquisition (S1), and testing (S2). Habituation to the testcage (single 5-min trial) took place on the fifth day of the weekduringMDMA treatment, and on the fourth day duringMPTPtreatment, 6 h after the last drug administration. Acquisitionwas performed the day after S0, by placing each mouse in thetest cage together with two identical copies of an object(familiar objects). Mice were left to freely explore the objectsfor 3 min. The testing phase took place 60 min after S1. Micewere exposed to one copy of the objects already presented inS1, plus an object that they had never experienced before(novel object).

Object exploration was defined as the mouse sniffing,gnawing, or touching the objects with its nose, whereassitting on and/or turning the objects around were not con-sidered exploratory behaviors. To avoid any olfactory cues,objects were thoroughly cleaned after each session. More-over, the combination of objects (familiar vs. novel) andtheir respective locations in the cage (right vs. left) werecounterbalanced, to prevent biased preferences for particu-lar objects and/or locations. The performance of the micewas videotaped, and the following parameters were evalu-ated: total amount of time spent by each mouse in exploringthe objects during S1 and S2 and percentage of time spentexploring the novel object over the total amount of timespent in exploring both objects (novel and familiar) duringS2 (Sawyer et al. 2012; Simola et al. 2008).

Spontaneous alternation behavior in a Y-maze

Evaluation of spontaneous alternation behavior in a Y-maze iswidely utilized to investigate spatial short-term memory andgeneral cognitive function in rodents (Maurice et al. 1994;Yamada et al. 1996). The apparatus used was made of blackPVC and had three equal arms (length 40 cm, width 11 cm,height 20.5 cm). The arms converged onto a central triangulararea, and the floor of the maze was covered with sawdust. Toavoid any olfactory cues, the maze was thoroughly cleanedand the sawdust was replaced in between each trial. Mice wereindividually placed in the central area and left to explore thewhole apparatus freely for a single 8-min trial, during whichtheir performance was videotaped. The percentage of sponta-neous alternations was calculated based on the sequence ofarm entries, as reported elsewhere (Yamada et al. 1996;Simola et al. 2008).

Statistics

Statistical analysis was performed with Statistica forWindows(StatSoft, Tulsa, OK, USA). Statistical significance was

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assessed by Student’s t test, one- or two-way analysis ofvariance (ANOVA), followed by Newman–Keuls post hoc testwhen appropriate. Three animals were excluded from the anal-ysis of behavioral tests because they did not perform the tasksassigned. Significance was set at p<0.05, and the results areexpressed as mean±S.E.M. for every analysis performed.

Results

Neuroinflammation in hippocampus and mPFC

Microglia

Vehicle+vehicle mice showed low levels of CD11b in boththe hippocampus and the mPFC (Fig. 2). Similarly, miceexposed to chronic MDMA (10 mg/kg twice a day/twice aweek) followed by vehicle administration did not show sig-nificant microglia activation. On the other hand, MPTP ad-ministration (20 mg/kg×4 administrations) to vehicle-pretreated mice elicited an elevation of microglia in the hip-pocampus compared with vehicle+vehicle and MDMA+ve-hicle mice. Finally, MPTP administration to MDMA-pretreated mice elicited a higher microglia stimulation in both

the hippocampus and the mPFC, compared with all the othergroups.

In the hippocampus, one-way ANOVA showed a signifi-cant effect of treatment (F3,25=9.19, p<0.01), and subsequentNewman–Keuls post hoc test revealed that the levels ofCD11b were higher in vehicle+MPTP mice and inMDMA+MPTP mice, compared with both vehicle+vehicleand MDMA+vehicle mice (Fig. 2(a and a’)).

In the mPFC, one-way ANOVA indicated a significanteffect of treatment (F3,16=6.89, p<0.01), and subsequentNewman–Keuls post hoc test showed that MDMA+MPTPmice exhibited CD11b levels significantly higher than thoseobserved in all the other experimental groups (Fig. 2(b and b’)).

Astroglia

Vehicle+vehicle mice displayed astroglial cells with highlybranched morphology, tiny processes, and a small body inboth the hippocampus and the mPFC (Fig. 3). Similarly, miceexposed to chronic MDMA (10 mg/kg twice a day/twice aweek) followed by vehicle did not show significant astrogliaactivation. On the other hand, MPTP administration(20 mg/kg×4 administrations) to vehicle-pretreated mice elic-ited an elevation in astroglia in the hippocampus compared

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Fig. 2 Effect of chronic MDMA and subsequent MPTP administrationon microglial activation in the hippocampus and mPFC. Late adolescentmice were treated with MDMA (10 mg/kg, twice a day/twice a week) for9 weeks and subsequently with MPTP (20 mg/kg×4 administrations).Representative sections and histograms of the hippocampus (a and a’)andmPFC (b and b’) immunostained for CD11b are shown. Experimental

groups: vehicle+vehicle (hippocampus n=9; mPFC n=5); MDMA+vehicle (hippocampus n=5; mPFC n=5); vehicle+MPTP (hippocampusn=7; mPFC n=5); and MDMA+MPTP (hippocampus n=8; mPFC n=5). Scale bar 50 μm. *P<0.05, **P<0.001 vs. vehicle+vehicle;#P<0.05, ##P<0.005 vs. MDMA+vehicle; ^^P<0.001 vs. vehicle+MPTP, by Newman–Keuls post hoc test

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with vehicle+vehicle mice. Moreover, MPTP administrationto MDMA-pretreated mice elicited a higher elevation in mi-croglia in the hippocampus and the mPFC compared with allthe other groups.

In the hippocampus, one-way ANOVA revealed asignificant effect of treatment (F3,34=9.42, p<0.01),and subsequent Newman–Keuls post hoc test showedthat the number of GFAP-positive cells was higher invehicle+MPTP mice compared with vehicle+vehiclemice, and in MDMA+MPTP mice compared with allthe other experimental groups (Fig. 3(a and a’)).

In the mPFC, one-way ANOVA showed a significant effectof treatment (F3,16=10.09, p<0.01), and subsequent New-man–Keuls post hoc test revealed that the number of GFAP-positive cells was higher in MDMA+MPTP mice, comparedwith all the other experimental groups (Fig. 3(b and b’)).

Behavioral tests

Novel object recognition (NOR) task

No significant modifications in NOR performance wereobserved during chronic administration (9 weeks) of

MDMA (10 mg/kg, twice a day/twice a week), comparedwith vehicle administration. Two-way ANOVA revealedno significant effect of chronic treatment (F1,29=0.04, p=0.84), no significant effect of time (F2,58=1.09, p=0.34),and no significant chronic treatment×time interaction(F2,58=0.51, p=0.34). However, when re-evaluated 2 weeksafter treatment discontinuation, MDMA-treated miceshowed an impairment in NOR performance comparedwith vehicle-treated mice, as shown by Student’s t test(df=32, t=4.10, p<0.01) (Fig. 4a).

When NOR performance was re-evaluated in the samemice 24 h after the last injection of MPTP (20 mg/kg×4administrations), one-way ANOVA revealed a significant ef-fect of treatment (F1,31=10.3, p<0.01). Subsequent Newman–Keuls post hoc test indicated that both MDMA+vehicle andvehicle+MPTP mice had an impaired NOR performance,compared with vehicle+vehicle mice, and that MDMA+MPTP mice had a poorer NOR performance compared withall the other experimental groups (Fig. 4b). In all the NORexperiments performed, no significant differences in the totalamount of time spent exploring the objects during either S1 orS2 were observed among the various experimental groups(data not shown).

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Fig. 3 Effect of chronic MDMA and subsequent MPTP administrationon astroglial activation in the hippocampus and mPFC. Late adolescentmice were treated with MDMA (10 mg/kg, twice a day/twice a week) for9 weeks and subsequently with MPTP (20 mg/kg×4 administrations).Representative sections and histograms of the hippocampus (a and a’)and mPFC (b and b’) immunostained for GFAP are shown. Experimental

groups: vehicle+vehicle (hippocampus n=9; mPFC n=5); MDMA+vehicle (hippocampus n=10; mPFC n=5); vehicle+MPTP (hippocam-pus n=9; mPFC n=5); and MDMA+MPTP (hippocampus n=10; mPFCn=5). Scale bar 50 μm. *P<0.05; **P<0.001 vs. vehicle+vehicle;##P<0.005 vs. MDMA+vehicle; ^P<0.05; ^^P<0.01 vs. vehicle+MPTP,by Newman–Keuls post hoc test

Psychopharmacology

Spontaneous alternation behavior in a Y-maze

No significant changes in spontaneous alternation be-havior were observed during chronic treatment withMDMA (10 mg/kg, twice a day/twice a week), com-pared with chronic vehicle administration (Fig. 5a).Two-way ANOVA revealed no significant effect of re-peated treatment (F1,29=1.8, p=0.19), no significant ef-fect of time (F2,58=1.24, p=0.30), and no significantrepeated treatment× time interaction (F2,58=1.2, p=0.31). Similar results were observed when mice werereevaluated 2 weeks after MDMA discontinuation, asshown by Student’s t test (df=32, t=0.33, p=0.74).Moreover, administration of MPTP (20 mg/kg×4 admin-istrations) did not affect spontaneous alternation behav-ior measured 48 h after the last MPTP injection, asshown by one-way ANOVA (F1,31=0.08, p=0.97)(Fig. 5b). Finally, mice treated with MDMA, MPTP,or their combination performed a similar number ofarm entries, compared with control mice (data notshown).

Discussion

Several lines of evidence have accumulated to suggestthat MDMA has neuroinflammatory properties and thatMDMA can amplify the effects elicited by other sub-stances that are toxic to the brain. The results of thisstudy substantiate and extend the current evidence andprevious findings from our group (Costa et al. 2013) bydemonstrating that chronic administration of MDMA tomice during adolescence exacerbates the neuroinflamma-tion elicited in the hippocampus and mPFC by MPTP, aneurotoxin relevant for humans that induces a syndromereproducing the features of PD (Langston et al. 1983),when administered at adulthood. Importantly, the presentinvestigation demonstrates that noxious MDMA–MPTPinteractions extend beyond motor areas and may havean influence on behavior, as revealed by the presence ofcognitive impairment in mice exposed to bothsubstances.

The exacerbation of neuroinflammation that can stem fromthe interactions between MDMA and other psychoactive

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Fig. 4 Effect of chronic MDMA and subsequent MPTP administrationon the performance of mice in the NOR task. Late adolescent mice weretreated with MDMA (10 mg/kg, twice a day/twice a week) for 9 weeksand subsequently with MPTP (20 mg/kg×4 administrations). a Repre-sentative histograms of NOR performance measured on weeks 1, 4, and 9of MDMA treatment and 2 weeks after MDMA discontinuation. b

Representative histograms of NOR performance measured 24 h afterthe last injection ofMPTP. Experimental groups:MDMA (n=18); vehicle(n=18); vehicle+vehicle (n=9); MDMA+vehicle (n=10); vehicle+MPTP (n=9); and MDMA+MPTP (n=8). **P<0.005 vs. vehicle+ve-hicle; #P<0.05 vs. MDMA+vehicle; ^P<0.05 vs. vehicle+MPTP, byStudent’s t test or Newman–Keuls post hoc test

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substances has recently attracted a great deal of attention (Costaet al. 2013; Hernandez-Rabaza et al. 2010; Izco et al. 2007;Khairnar et al. 2010; Ros-Simò et al. 2012, 2013). Thus, it isbecoming increasingly clear that MDMA should be regarded asharmful, not only because its use may have implications fordrug dependence (Fernandez-Serrano et al. 2011; Smirnov et al.2013) and may affect heart function and body temperatureregulation (McNamara et al. 2006, 2007; Parrott 2012), butalso because MDMA could function as a “primer” for laterbrain insults. In this regard, the present study acquires particularrelevance, as it provides further evidence that MDMA exacer-bates the neuroinflammatory effects of a toxin that induces PD.Remarkably, the results obtained demonstrate that such anexacerbation takes place in the limbic and cortical regions; this,together with previous evidence obtained in motor areas (Costaet al. 2013), suggests that the deleterious influence of MDMAon neuroinflammation is widespread.

The amplification of MPTP-mediated neuroinflammation inmice pretreated with MDMA during adolescence appeared to beparticularly marked in the mPFC, involving both microglia andastroglia. Moreover, it is noteworthy that MPTP alone did notsignificantly stimulate neuroinflammation in the mPFC. Thislatter finding deserves consideration, since it suggests that theneurochemical modifications induced by chronic exposure toMDMA may prime the brain for the effects of later noxiousstimuli. At variance with these results, only MPTP-inducedastrogliosis, but not microgliosis, was exacerbated in the hippo-campus of MDMA-pretreated mice, compared with MPTPalone. Such an amplification appeared to be less pronouncedthan the one observed in the mPFC, and in addition, MPTP

elicited both astrogliosis and microgliosis in the hippocampuswhen given alone. The lack of neuroinflammatory effects ofMDMA observed in this study may appear to be in contrast withprevious data obtained in experimental animals showing thatMDMA elicits neuroinflammation in the hippocampus (Buscetiet al. 2008; Straiko et al. 2007). This discrepancy could beexplained, in the first instance, considering that in the presentstudy immunohistochemical evaluation was performed 3 weeksafter the last MDMA administration, and therefore, the acuteneuroinflammatory effects of MDMA, which have a short-timewindow (Granado et al. 2008), may have faded at a later time.Moreover, the differences in doses and protocols of MDMAadministration between the present and previous studies shouldbe taken into consideration. As for MPTP, this study confirmsprevious evidence reporting the neuroinflammatory effects ofMPTP in the hippocampus (Kokovay and Cunningham 2005;Sy et al. 2010; Wang et al. 2009), which could be explained bythe presence of a very high density of binding sites for 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite derived fromMPTP oxidation, in this region (Del Zompo et al. 1992).

Different mechanisms could underlie the exacerbation ofMPTP-mediated neuroinflammatory effect observed inMDMA-pretreated mice. MDMA has been reported to inhibitthe CYP2D6 isoform of the cytochrome P450 (O’Mathùnaet al. 2008), which has been shown to convert MPTP intonontoxic metabolites (Herraiz et al. 2006; Mann and Tyndale2010). Moreover, MDMA has been demonstrated to inhibitmitochondrial complex I in mice (Puerta et al. 2010), andinhibition of mitochondrial complex I is also at the basis ofMPTP-mediated inflammatory effects (Fallon et al. 1997).

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Spontaneous alternation during MDMA treatment and after washout

Spontaneous alternation after MPTP administration

Fig. 5 Effect of chronic MDMAand subsequent MPTPadministration on thespontaneous alternation behaviorof mice in a Y-maze. Late ado-lescent mice were treated withMDMA (10 mg/kg, twice a day/twice a week) for 9 weeks andsubsequently with MPTP (20 mg/kg×4 administrations). a Repre-sentative histograms of spontane-ous alternation behavior mea-sured on weeks 1, 4, and 9 ofMDMA treatment and 2 weeksafter MDMA discontinuation. bRepresentative histograms ofspontaneous alternation behaviormeasured 24 h after the last in-jection of MPTP. Experimentalgroups: MDMA (n=19); vehicle(n=17); vehicle+vehicle (n=8);MDMA+vehicle (n=10); andvehicle+MPTP (n=9); MDMA+MPTP (n=9)

Psychopharmacology

Another important mechanism may involve oxidative stress,based on previous studies suggesting that prolonged exposureto MDMA may result in the intracellular generation of prooxi-dant species (Halpin et al. 2013; Perfeito et al. 2012). In thisconnection, it is noteworthy that MDMA may promote therelease of dopamine (DA) (Cadoni et al. 2005) which canundergo auto-oxidation, and in turn generate free radicals andROS (Barbosa et al. 2012; Hondebrink et al. 2011), eventuallyleading to the exacerbation of the neuroinflammatory phenome-na triggered byMPTP, which itself acts by elevating the levels offree radicals and ROS. This latter mechanism could be particu-larly relevant to the results of this study, as it might justify whyMDMA pre-exposure was associated with a more marked exac-erbation of MPTP-neuroinflammation in the mPFC, which ishighly enriched with dopaminergic terminals (Pycock et al.1980), than the hippocampus, where dopaminergic innervationis less dense (Verney et al. 1985). In addition, it is interesting tomention that our previous study (Costa et al., 2013) observedhow MPTP administration to MDMA-pretreated mice elicited amore marked degeneration of dopaminergic neurons in the SNc,compared with either drug alone. This finding may acquirerelevance to the present results, as a significant percentage ofdopaminergic projections to the mPFC originate in the centralregion of the SNc (Albanese et al. 1986; Maurice et al. 1999;Middleton and Strick 2002). Therefore, it can be hypothesizedthat a degeneration of neurons projecting to the mPFC maycontribute, at least in part, to the onset and persistence of theneuroinflammatory changes observed in this study.

In addition to neuroinflammatory changes, mice treated withMDMAduring adolescence displayed a persistent impairment inNOR performance after treatment discontinuation, and MDMApretreatment was found to further aggravate cognitive impair-ment induced by MPTP. These results appear to be of greatrelevance since, despite the ever-increasing use of MDMA inhumans, scarce information is available on the long-term effectson cognition elicited by the consumption of this drug duringadolescence. Importantly, the impairment in NOR performanceobserved in this study appears to be clearly attributable to deficitsin cognitive function. In fact, no differences were found betweenMDMA- and vehicle-treated mice as for the total amount of timespent in object exploration during the two phases of the NORtask. This indicates that the regimen ofMDMAused in this studyis devoid of nonspecific effects on object exploration. Moreover,MDMA did not seem to alter the ability of mice to discriminatebetween different objects. In fact, mice could recognize novelobjects at different time points during the MDMA treatment, buteventually lost this ability when reevaluated at 2 weeks aftertreatment discontinuation, which would suggest the onset ofprogressive and long-term MDMA effects. Both the hippocam-pus and the mPFC have been shown to be involved in itemrecognition in rodents (Dalley et al. 2004; Driscoll et al. 2003;Ragozzino et al. 1999); therefore, it is feasible that the impair-ment in NOR performance observed here may be linked to the

neuroinflammatory effects elicited by MDMA and MPTP inthese areas. Remarkably, support for the existence of a causallink between neuroinflammation and cognitive impairmentcomes from previous studies which have employed the systemicor intrahippocampal injection of lipopolysaccharide in rodents.Lipopolysaccharide has been shown not only to stimulate theexpression of inflammation-related molecules, including GFAP,inducible nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, IL-6, and IL-1β (Herber et al. 2006; Jain et al. 2002; Pang et al.2006; Sparkman et al. 2006), but also to engender cognitiveimpairment. Furthermore, methamphetamine, a highly addictivepsychostimulant, the abuse of which in humans is often associ-ated with neurocognitive impairment (Nestor et al. 2011; Northet al. 2013), is known to induce neuroinflammatory responses inmice that are characterized by an upregulation of the levels ofGFAP and CD11b, tumor necrosis factor (TNF)-α, and TNF-receptor 1 protein (Gonçalves et al. 2010), and by dopaminergictoxicity (Sonsalla et al. 1989). In the present study, cognitiveimpairment was narrowed to NOR, while no deficits were de-tectable in spontaneous alternation behavior. This lack of effectcould be observed both during MDMA treatment and followingMPTP administration, and no exacerbation of MPTP effects waspresent inMDMA-treatedmice. These results could be explainedin light of previous studies that have shown how spontaneousalternation behavior may be relatively resistant to noxious braininsults that impair NOR performance, suggesting a differentsensitivity of these two tasks to cognitive function disruption(Moriguchi et al. 2012; Simola et al. 2008). Moreover, it is worthmentioning that MPTP has been demonstrated to scarcely affectalternation behavior in mice (Tanila et al. 1998), which couldjustify the lack of an impairment in spontaneous alternationfollowing MPTP observed here.

The present study, encompassing both neurochemical andbehavioral findings, may significantly contribute to the elucida-tion of the interactions between MDMA and substances that aretoxic to the brain, and appear to be also of potential relevance tohumanMDMA consumption. Thus, a major conclusion that canbe drawn from the data obtained is that the detrimental effects ofMDMA, even if subtle, appear to be long lasting, since theycould be detected after treatment discontinuation. In addition,MDMA was administered during adolescence, reproducing acondition similar to that observed in humans, where MDMA islargely consumed by adolescents and young adults (Singer et al.2004). In this regard, it is noteworthy that adolescence is a criticalperiod for brain maturation and remodeling, and that the adoles-cent brain is particularly susceptible to the effects of exogenoussubstances, including drugs of abuse (Cadoni et al. 2013; Daza-Losada et al. 2009; Sisk and Zehr 2005; Spear 2000). Moreover,it has to be emphasized that DA plays a key role in synaptogen-esis (Fasano et al. 2008; Goffin et al. 2010; Sisk and Zehr 2005),and it may greatly influence brain remodeling during adoles-cence. Therefore, the intake of a substance, such asMDMA, thatcan alter DA transmission (Spear 2000) may severely impact this

Psychopharmacology

process, leading to long-lasting effects, which could eventuallyrender the brain more sensitive to toxic insults later in life. Inparticular, the abuse of MDMA, and other amphetamine-relateddrugs, during adolescence may not only be critical for the devel-opment of addiction, but also create the basis for a worsening ofcognitive function, in agreement with that observed by severalstudies in adult MDMA consumers (Becker et al. 2013; Bollaet al. 1998; Bosch et al. 2013; Daumann et al. 2004, 2005;Moeller et al. 2004; Parrott and Lasky 1998).

In conclusion, by demonstrating that mice chronicallytreated with MDMA during adolescence display an exacerba-tion of neuroinflammation and cognitive impairment inducedby MPTP, the present results underline the importance ofexploring the long-term effects of amphetamine-related drugsto clarify their potential as risk factors for the development ofneurological and cognitive problems later in life.

Acknowledgments This study was supported by funds from RegioneAutonoma della Sardegna (Legge Regionale 7 Agosto 2007, N.7,annualità 2008 and 2010). Dr. Giulia Costa gratefully acknowledges theSardinian Regional Government for financial support (P.O.R. SardegnaF.S.E. Operational Programme of the Autonomous Region of Sardinia,European Social Fund 2007–2013–Axis IV Human Resources, Line ofActivity l.3.1 “Finanziamento ai corsi di dottorato finalizzati allaformazione di capitale umano altamente specializzato”). Dr. NicolaSimola gratefully acknowledges the Sardinian Regional Governmentfor financial support (P.O.R. Sardegna F.S.E. Operational Programme ofthe Autonomous Region of Sardinia, European Social Fund 2007–2013 –Axis IV Human Resources, Objective l.3, Line of Activity l.3.1 “Avvisodi chiamata per il finanziamento di Assegni di Ricerca”).

The authors are grateful to Prof. Antonio Plumitallo and Mr. RenatoMascia, Department of Life and Environmental Sciences, University ofCagliari, for their help with the MDMA synthesis.

Conflicts of interest Nothing to report.

Funding agencies This study was supported by funds from theRegione Autonoma della Sardegna (Legge Regionale 7 Agosto 2007,N.7, annualità 2008 and 2010). Dr. Giulia Costa and Dr. Nicola Simolawere supported by funds fromRegione Autonoma della Sardegna (P.O.R.FSE 2007–2013).

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