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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Modafinil improves methamphetamine-induced object recognitiondeficits and restores prefrontal cortex ERK signaling in mice

Betina González a, Mariana Raineri a, Jean Lud Cadet c, Edgar García-Rill d,Francisco J. Urbano b, Veronica Bisagno a,*

a Instituto de Investigaciones Farmacológicas (Universidad de Buenos Aires e Consejo Nacional de Investigaciones Científicas y Técnicas),Ciudad Autónoma de Buenos Aires, Junín 956, Piso 5, C1113 Buenos Aires, Argentinab Laboratorio de Fisiología y Biología Molecular, Instituto de Fisiología, Biología Molecular y Neurociencias (Universidad de Buenos Aires e Consejo Nacionalde Investigaciones Científicas y Técnicas), Ciudad Autónoma de Buenos Aires, Buenos Aires, ArgentinacMolecular Neuropsychiatry Research Branch, NIH/NIDA Intramural Research Program, Baltimore, MD, United StatesdCenter for Translational Neuroscience, Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock,AR, United States

a r t i c l e i n f o

Article history:Received 10 October 2013Received in revised form29 January 2014Accepted 4 February 2014

Keywords:ModafinilMethamphetamineNoveltyERKPrefrontal cortex

* Corresponding author. Instituto de Investigacio(CONICET-UBA), Junín 956, 5, Ciudad de Buenos AiresTel.: þ54 11 4961 6784; fax: þ54 11 4963 8593.

E-mail addresses: vbisagno@ffyb.uba.ar, v_bisagno

http://dx.doi.org/10.1016/j.neuropharm.2014.02.0020028-3908/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: González,prefrontal cortex ERK signaling in mice, Neu

a b s t r a c t

Chronic use of methamphetamine (METH) leads to long-lasting cognitive dysfunction in humans and inanimal models. Modafinil is a wake-promoting compound approved for the treatment of sleeping dis-orders. It is also prescribed off label to treat METH dependence. In the present study, we investigatedwhether modafinil could improve cognitive deficits induced by sub-chronic METH treatment in mice bymeasuring visual retention in a Novel Object Recognition (NOR) task. After sub-chronic METH treatment(1 mg/kg, once a day for 7 days), mice performed the NOR task, which consisted of habituation to theobject recognition arena (5 min a day, 3 consecutive days), training session (2 equal objects, 10 min, day4), and a retention session (1 novel object, 5 min, day 5). One hour before the training session, mice weregiven a single dose of modafinil (30 or 90 mg/kg). METH-treated mice showed impairments in visualmemory retention, evidenced by equal preference of familiar and novel objects during the retentionsession. The lower dose of modafinil (30 mg/kg) had no effect on visual retention scores in METH-treatedmice, while the higher dose (90 mg/kg) rescued visual memory retention to control values. We alsomeasured extracellular signal-regulated kinase (ERK) phosphorylation in medial prefrontal cortex(mPFC), hippocampus, and nucleus accumbens (NAc) of METH- and vehicle-treated mice that receivedmodafinil 1 h before exposure to novel objects in the training session, compared to mice placed in thearena without objects. Elevated ERK phosphorylation was found in the mPFC of vehicle-treated mice, butnot in METH-treated mice, exposed to objects. The lower dose of modafinil had no effect on ERKphosphorylation in METH-treated mice, while 90 mg/kg modafinil treatment restored the ERK phos-phorylation induced by novelty in METH-treated mice to values comparable to controls. We foundneither a novelty nor treatment effect on ERK phosphorylation in hippocampus or NAc of vehicle- andMETH-treated mice receiving acute 90 mg/kg modafinil treatment. Our results showed a palliative role ofmodafinil against METH-induced visual cognitive impairments, possibly by normalizing ERK signalingpathways in mPFC. Modafinil may be a valuable pharmacological tool for the treatment of cognitivedeficits observed in human METH abusers as well as in other neuropsychiatric conditions.

This article is part of a Special Issue entitled ‘CNS Stimulants’.� 2014 Elsevier Ltd. All rights reserved.

nes Farmacológicas, ININFAC1113AAD, CABA, Argentina.

@yahoo.com (V. Bisagno).

B., et al., Modafinil improvesropharmacology (2014), http

1. Introduction

Methamphetamine (METH) is a psychostimulant with a highpotential for abuse and addiction. Repeatedexposure toMETHcausesabnormal changes in neurotransmitter activity involved in learning,reward, and executive function (Bamford et al., 2008; Feltenstein andSee, 2008). METH also alters neuronal plasticity in brain regions that

methamphetamine-induced object recognition deficits and restores://dx.doi.org/10.1016/j.neuropharm.2014.02.002

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mediate cognition and motivation (Kauer and Malenka, 2007;Robinson and Kolb, 2004). METH addiction generally begins withrecreational use and progresses over time into a compulsive andchronically relapsing disorder, accompanied by psychiatric symp-toms including hallucinations and delusions, as well as long-termcognitive deficits (Scott et al., 2007). METH-dependent individualsexhibit high rates of cognitive dysfunction in several neuropsycho-logical domains that include sustained attention, episodic memory,information processing, and impulse control (Monterosso et al.,2005; Nordahl et al., 2003; Simon et al., 2010; Morgan et al., 2012).These cognitive deficitsmight undermine efforts byMETH addicts tostop or reduce METH use and negatively affect the outcome oftreatment (Vocci andAppel, 2007). Given the potential links betweencognition and treatment outcome inMETH dependence, therapeuticapproaches that improve cognitive function may be quite promisingin the management of METH addiction.

Modafinil (Provigil) is a psychostimulant and cognitiveenhancer drug, approved by the U.S. Food and Drug Administrationfor treating narcolepsy and other sleep disorders. Different studiesshowed that modafinil cognitive-enhancing properties improvedoutcome in the treatment of pathologic gamblers (Zack and Poulos,2009), alcoholics (Schmaal et al., 2013), and patients suffering fromother neuropsychiatric conditions (Scoriels et al., 2013). The use ofmodafinil as a treatment for cocaine and METH dependence re-mains inconclusive, with studies showing positive outcomes(Dackis et al., 2005; McGaugh et al., 2009) and studies showingpromising but yet non-significant results in reducing drug use(Dackis et al., 2012; Shearer et al., 2009; Heinzerling et al., 2010).For both cocaine and METH users, modafinil was efficacious inimproving several domains of cognitive and executive functions(Kalechstein et al., 2013; Ghahremani et al., 2011; Kalechstein et al.,2010; Hester et al., 2010; Finke et al., 2010).

Modafinil’s mechanism of action, although somewhat poorlyunderstood, appears to involve multiple neurotransmitter systems.For example, modafinil can act as a weak DA transporter (DAT)inhibitor that increases extracellular dopamine (DA) levels (Mereuet al., 2013). Modafinil influences GABAergic, glutamatergic,noradrenergic, serotoninergic, histaminergic, and orexinergic sys-tems (for a review see Minzenberg and Carter, 2008; Scoriels et al.,2013). In addition, modafinil enhances electrotonic coupling byincreasing the effectiveness of gap junctions between neurons(Urbano et al., 2007; Garcia-Rill et al., 2007). We also demonstratedthat modafinil can protect against METH toxicity (Raineri et al.,2011, 2012). Specifically, modafinil was able to prevent METH-induced toxic effects that included DA depletion and reductionsin tyrosine hydroxylase (TH) and DAT levels in the striatum (Raineriet al., 2011). Furthermore, modafinil also attenuatedMETH-inducedhyperthermia, glial activation, and increased expression of pro-apoptotic proteins (Raineri et al., 2012).

Compared to classical psychostimulants such as cocaine oramphetamine, the sites of action and behavioral effects of modafinilappear to be different (Mereu et al., 2013). Modafinil showed lowerliability to abuse and lower risk of adverse effects on organ systemsincluding the cardiovascular system (Minzenberg and Carter,2008). Clinically relevant modafinil doses can robustly activatefronto-cortical areas involved in higher cognitive functions and anetwork of pro-arousing areas, which provide a plausible substratefor the wake-promoting and pro-cognitive effects of the drug(Gozzi et al., 2012). Of relevance to the present study, METH-dependent subjects showed a greater effect of modafinil on brainactivation in bilateral insula/ventrolateral prefrontal cortex andanterior cingulate cortices than control participants, suggestingthat modafinil improves learning in METH-dependent participantsby enhancing neural function in those regions (Ghahremani et al.,2011).

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The intracellular signaling pathways that mediate modafinilactions in fronto-cortical areas remain unknown. A potentialcandidate is the mitogen-activated protein kinase extracellularsignal-regulated kinase (MAPK-ERK) cascade. The ERK1/2 pathwayplays a critical role in memory function under physiological andpathological conditions (Mizoguchi et al., 2004; Kamei et al., 2006;Nagai et al., 2007; Cammarota et al., 2008). ERK1/2 signalingpathway linked to dopamine D1 receptors (Valjent et al., 2000;Zanassi et al., 2001) is involved in METH-associated contextualmemory in rats (Mizoguchi et al., 2004). Additionally, it has beendemonstrated that repeated METH treatment in mice inducedcognitive impairment in a novel object recognition test, which wasassociated with deficits of the ERK1/2 pathway in the prefrontalcortex (PFC) (Kamei et al., 2006).

As it was mentioned above, there is only limited information onthemechanisms bywhichmodafinil improves cognition in patientswith addictive behaviors in terms of underlying neural substrates.Therefore, in the present study, we designed experiments aimed attesting modafinil’s ability to improve cognitive deficits induced bysub-chronic METH treatment in mice. We used a novel objectrecognition (NOR) task, which is similar to visual recognition testswidely used in subhuman primates (Ennaceur, 2010), and is sen-sitive to METH-induced cognitive impairments (Bisagno et al.,2002; Kamei et al., 2006; Reichel et al., 2011). The NOR task eval-uates the rodents’ ability to recognize a novel object in the envi-ronment, and discrimination and memory performance is obtainedupon identification of familiarity and novelty (Antunes and Biala,2012). So, we also examined how modafinil and METH differen-tially modulated fronto-cortical phosphorylation of the MAPK iso-forms ERK1/ERK2 following exposure to novelty.

2. Materials and methods

2.1. Animals

C57BL/6 male mice (2e3 month-old) from the School of Exact and NaturalSciences of the University de Buenos Aires (UBA) were housed in a light andtemperature-controlled room (12-h light/dark cycle, 22 �C), and were given freeaccess to laboratory chow and tap water. Principles of animal care were followed inaccordance with ‘‘Guidelines for the Care and Use of Mammals in Neuroscience andBehavioral Research’’ (National Research Council, 2003) and approved by Uni-versidad de Buenos Aires authorities (Protocol Number: A5801-01) using OLAWandARENA directives (NIH, Bethesda, USA).

2.2. Pharmacological reagents

Drugs were purchased from either Sigma (St. Louis, MO) or Tocris (Ellisville,MO). Modafinil (racemic mixture of R- and S-enantiomers) was generously donatedby Laboratorios Beta S.A. (Argentina).

2.3. Drug treatments

(þ)-Methamphetamine hydrochloride (Sigma, St Louis, MO) was administeredsubcutaneously (sc) once a day for 7 days (1 mg/kg, calculated as free base, dissolvedin sterile saline solution). The METH regimen used in this study was performedaccording to studies by Kamei et al. (2006). Four days after the last METH injection,modafinil (30 or 90 mg/kg, dissolved in DMSO-Arabic gum 5% in sterile saline so-lution) was injected and 1 h later mice were subjected to behavioral analysis (NovelObject Recognition task or Novelty exposure, Fig. 1A and B). Vehicle groups receivedthe same volume of sterile saline and DMSO/Arabic gum/saline. Drugs were injectedat a volume of 10 ml/kg of body weight.

2.4. Novel Object Recognition (NOR) task

The NOR task was adapted according to previously reported methods (Kameiet al., 2006). The NOR task evaluates the rodents’ ability to recognize a novel ob-ject in the environment. Basically, in the NOR task there are no positive or negativereinforcers, and this methodology assesses the natural preference for novel objectsdisplayed by rodents (Ennaceur, 2010). The task procedure consists of three phases:habituation, training, and a test phase. In the habituation phase, each animal isallowed to freely explore the arena in the absence of objects. The animal is thenremoved from the arena and placed in its cage. During the training phase, a singleanimal is placed in the arena containing two identical sample objects (A þ A) for5 min. The experimental context is not drastically different during the training andthe test phase. After a retention interval begins the test phase, and the animal is

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returned to the arenawith two objects, one is identical to the sample and the other isnovel (A þ B).

In our experiments, exploration occurred in an open-top arena (40 cm3) made ofplexiglass, with the floor covered with clean woodchip bedding. Testing was doneduring the light phase (8 AMe8 PM), in a sound-attenuated room, with dimmedillumination (40 W). Mice were individually habituated to the box for 5 min duringthree consecutive days after the last METH injection, in the absence of objects(Fig. 1B: habituation sessions, withdrawal days 1e3). On the fourth day after METH,two identical objects were symmetrically fixed to the floor of the box, 8 cm from oneof the walls, and each mouse was allowed to explore the box for 10 min (Fig. 1B:training session, withdrawal day 4). Objects were: golf balls, plastic pipes (3 cmdiameter, 8 cm high) and plastic cubes (4 cm3), all of which were similar in size butdifferent in color, shape and brightness. Sets of objects were chosen based on pre-liminary experiments that indicated that they were similarly preferred. Objects werewashed with 40% ethanol solution between trials. Following a 24-h delay, mice wereplaced back in the box for 5 min where one of the familiar objects was replaced by anovel object (Fig. 1B: retention session, withdrawal day 5). The positions of the ob-jects in the test and the objects used as novel or familiar were counterbalanced be-tween the animals in each group and between the control and drug-treated groups.All sessions were recorded and analyzed with Ethovision XT 7.0 tracking software(Noldus, The Netherlands), using nose point-tail base detection. The percentage ofexploratory preference (%EP) was calculated as exploration time of the novel object(TN) divided by the total exploration time of both novel (TN) and familiar (TF) objects[%EP ¼ TN/(TN þ TF) * 100]. Similarly, this calculation can be applied when bothobjects are identical, in the training session phase, but here the mathematic formulawill be exploration time of the right object (TR) divided by the total exploration timeof both right (TR) and left (TL) objects [%EP ¼ TR/(TR þ TL) * 100].

2.5. Novelty effect

In a separate set of experiments, mice were individually habituated to objectrecognition arena for 5 min during three consecutive days after the last METH in-jection (in the absence of objects). On theMETHwithdrawal day 4, mice were placedin the arena for 10 min with objects (Novelty) or without objects (Control) (Fig. 1B),and then immediately euthanized. Brains were quickly removed and brain areaswere dissected out on an ice-cold plate. Each tissue sample was frozen and storedat �70 �C until processed.

2.6. Exploratory activity

We investigated the effect of early METH withdrawal on exploratory activity.Spontaneous locomotor activity was recorded during habituation sessions to theobject recognition arena using Ethovision XT 7.0 tracking software (Noldus, TheNetherlands). We analyzed locomotor activity (distance traveled) and time spent inthe center area of the arena during the 5-min habituation sessions. The center areawas defined as one half the total area of the arena.

2.7. Western blot

We measured ERK and phosphorylated ERK (pERK) protein expression byWestern blot in brain areas of mice subjected to the novelty effect. Brains were

Fig. 1. Schematic representations of the experimental treatments and behavioral analysis.1 mg/kg, sc, once a day for 7 consecutive days. On day 4 after sub-chronic treatment, mice reMETH withdrawal mice were habituated to the object recognition arena 5 min a day for 3(NOR) task or were evaluated for the Novelty effect. For the NOR task, mice performed a traiand 24 h later (day 5) performed a 5 min retention session, where one of the familiar objectsNovelty session where they were allowed to explore two equal novel objects and euthanizedsame procedure was performed.

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removed rapidly after Novelty and Control sessions and brain sections weredissected out and stored at �70 �C. Tissue homogenates were prepared in a solutioncontaining 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X100, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 mM PMSF, 5 mg/ml leupeptin, and 5 mg/ml aprotinin. Afterremoval of cell debris by centrifugation, the protein concentration of the cell lysatewas determined. The homogenates were combined with loading buffer containing4% SDS, 20% glycerol, 10% b-mercaptoethanol, 125 mM Tris, (pH 6.8), and boiled at100 �C for 5 min. Protein samples (50 mg) were separated by 12.5% SDS-PAGE, andthe separated proteins transferred to a PVDF membrane. Immunoblotting wasperformed using a mouse monoclonal antibody to pERK1/2 (1:500, E-4 sc-7373Santa Cruz Biotechnology), then membranes were stripped and reprobed withpolyclonal rabbit antibody to total ERK2 (1:2000, C-14 sc-154 Santa Cruz Biotech-nology). Because there was no change in the levels of total ERK, values of pERK werenormalized to the values of total ERK. Immune complexes were detected with anti-mouse and anti-rabbit secondary antibodies and chemiluminescence reagents(Amersham, NJ, USA). Bands were quantified using ImageJ (NIH).

2.8. Statistical analysis

InfoStat software (www.infostat.com.ar) was used for statistical comparisons.Statistics were performed using either one-way (treatment) or two-way (noveltyand treatment) ANOVA followed by Bonferroni post hoc tests or planned contrasts.Data were transformed when required. Kruskal Wallis ANOVA on Ranks was per-formed when data did not comply with the assumptions of parametric tests. For theanalysis of exploratory activity during habituation sessions, repeated measures two-way ANOVA followed by Bonferroni was performed. Differences were consideredsignificant if p < 0.05.

3. Results

3.1. Effects of modafinil and methamphetamine on exploratoryactivity and object recognition memory

3.1.1. Effect of methamphetamine withdrawal on exploratoryactivity

As shown in Fig. 1A, mice were treated with a chronic METH orvehicle protocol (METH 1 mg/kg/day for 7 consecutive days). Next,mice were placed in an object recognition arena 5-min a day for 3consecutive days, as part of the habituation sessions for the NORtask. We recorded these habituation sessions and analyzedexploratory activity during METH withdrawal, measuring locomo-tion and time spent in the center of the arena (Fig. 2). Repeatedmeasures two-way ANOVA for locomotion showed significant dayeffect [F(2,50) ¼ 21.75, p < 0.001], but no treatment or interactioneffect (Fig. 2A). Bonferroni post hoc test showed that on day 1 ofwithdrawal both METH- and vehicle-treated mice manifested

A) Male C57BL/6 mice were subjected to sub-chronic treatment with saline or METHceived vehicle or modafinil (MOD) 90 or 30 mg/kg, ip, 1 h before behavioral analysis. B)consecutive days. On withdrawal day 4, mice performed the Novel Object Recognitionning session in which they were allowed to freely explore two equal objects for 10 min,was replaced by a novel object. To evaluate the Novelty effect, mice performed a 10 minimmediately. Control mice were placed in the arena in the absence of objects and the

methamphetamine-induced object recognition deficits and restores://dx.doi.org/10.1016/j.neuropharm.2014.02.002

Fig. 2. Effect of chronic METH withdrawal on exploratory activity. Mice were treated with vehicle or METH (1 mg/kg, sc, once a day for 7 days), and onwithdrawal were placed in anopen-top arena in a 5 min habituation session for 3 consecutive days. A) Locomotion, *: Day 1 different from Day 2 and Day 3 (p < 0.05). B) Time in center, *: vehicle different fromMETH in Day 2. Exploratory activity during METH withdrawal was evaluated using Ethovision XT 7.0 tracking software (Noldus, Leesburg, Virginia). Repeated measures two-wayANOVA e Bonferroni. Values indicate mean � SEM (N ¼ 8e9).

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increased locomotion (p < 0.05), compared to day 2 and 3, sug-gesting habituation to the object recognition arena. Repeatedmeasures two-way ANOVA for time in center showed significanttreatment [F(1,50)¼ 5.14, p< 0.05], day [F(2,50)¼ 18.81, p< 0.001],and interactive [F(2,50) ¼ 3.69, p < 0.05] effects (Fig. 2B). Bonfer-roni post hoc test showed that on day 2 the Vehicle group dimin-ished the time spent in the center (p< 0.05), while theMETH groupdid not. On day 3 both groups spent similar amounts of timeexploring the center. These results indicate that, by the timetraining sessions of the NOR task had started, both METH andcontrol mice showed similar exploratory activity andwere similarlyhabituated to the object recognition arena.

3.1.2. Acute modafinil effects on methamphetamine withdrawal:object recognition memory

On day 4 of withdrawal, mice received acute vehicle, 30 or90 mg/kg modafinil dose and 1 h later performed the trainingsession of the NOR task. As expected, one way ANOVA found nosignificant differences in the exploratory preference for the trainingsessions after either 30 or 90 mg/kg modafinil (Fig. 3A and B). Theretention session was performed 24 h later, and both 30 mg/kg and90 mg/kg modafinil groups showed significant treatment effect(modafinil 30 mg/kg: Kruskal Wallis ANOVA on ranks [H ¼ 10.26,p < 0.05]; modafinil 90 mg/kg retention session: one way ANOVA-Bonferroni [F(3,70) ¼ 8.11, p < 0.001]). For both 30 and 90 mg/kggroups, post hoc tests showed impaired object recognitionmemoryin METH-treated mice, evidenced by similar preference of familiarand novel objects during the retention session of the NOR task(Fig. 3A and B). Acute 30 mg/kg modafinil did not improve objectrecognition deficits observed in METH-treated mice (Fig. 3A).Modafinil at a higher dose, 90 mg/kg, improved METH-inducedobject recognition deficits, evidenced by a significant increase inthe preference index in the METH-MOD group compared to theMETH group (p < 0.05), to values comparable to those of theVehicle group (Fig. 3B). Modafinil-treated subjects (for 30 and90 mg/kg) showed preference values similar to the Vehicle group,and significantly higher compared to those of the METH group(p< 0.05). Total time spent in object exploration during the trainingand retention sessions did not differ among groups for the twomodafinil doses (Fig. 3C and D). These results suggest that chronicMETH treatment did not have an effect on motivation and explo-ration, suggesting METH-induced memory impairment rather thana lack of interest in novel stimuli. We also measured locomotoractivity during training and retention sessions (Fig. 3E and F). Both30 mg/kg and 90 mg/kg modafinil groups showed significanttreatment effect in the training session (MOD 30 mg/kg: one wayANOVA-Bonferroni [F(3,38) ¼ 11.45, p < 0.001]; MOD 90 mg/kg:

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one way ANOVAeBonferroni [F(3,77) ¼ 66.47, p < 0.001]). Post hoctests showed no differences in locomotor activity of Vehicle- andMETH-treated mice, while the acutely modafinil-treated mice(MOD and METH-MOD groups) showed an increase in locomotoractivity compared to Vehicle and METH groups at both modafinildoses (p< 0.05). It is noteworthy that the 90mg/kg theMETH-MODgroup exhibited higher locomotor activity compared with mod-afinil group values (p < 0.05) (Fig. 3E). One way ANOVA found nosignificant differences in locomotion for the retention sessions ineither 30 or 90 mg/kg modafinil groups. In addition, modafinil-induced improvement of object recognition memory deficits afterMETHwas replicated in female mice (data not shown). We used thesame protocol previously described for male mice using modafinilat a dose that showed positive results, i.e. 90 mg/kg. Exploratorypreferences in retention session (%) were as follows: Vehicle:65.01 � 4.20 [a], MOD: 66.34 � 3.61 [a], METH: 44.91 � 8.41 [b],METH-MOD: 66.21 � 7.01 [a], one way ANOVAeBonferroni[F(3,24) ¼ 3.02, p < 0.05].

3.2. Effects of modafinil and methamphetamine locomotor activityand ERK phosphorylation induced by novelty

The results obtained in the NOR task showed that modafinil,given at a high 90 mg/kg dose before the first exposure to novelobjects, was able to enhance long-term memory retention byrestoring the novelty preference in a retention session performed24 h later. As mentioned above, the NOR task evaluates the ro-dents’ ability to recognize a novel object in the environment anddiscrimination and memory performance is obtained upon iden-tification of familiarity and novelty (Antunes and Biala, 2012).Novelty is a change from expected likelihood of an event on thebasis of both previous information and internal estimates ofconditional probabilities (Antunes and Biala, 2012). Animals canbe affected by a novel stimulus; i.e. novelty can change the ani-mals’ behavior, induce stress responses, and elicit approachbehavior (Bevins et al., 2002). Therefore, we conducted separateexperiments in mice subjected to METH withdrawal aimed atinvestigating novelty-induced changes on mPFC ERK phosphory-lation and exploratory activity with or without modafiniladministration.

3.2.1. Effect of acute modafinil on mice exposed to novel objectsduring methamphetamine withdrawal: locomotor activity

To asses novelty-induced locomotor activity, mice were placedin the arena with or without objects (Novelty and Control groups,respectively) for 10 min, and euthanized immediately (Fig. 1B). Werecorded Control and Novelty sessions and analyzed locomotor

methamphetamine-induced object recognition deficits and restores://dx.doi.org/10.1016/j.neuropharm.2014.02.002

Fig. 3. Effect of modafinil (MOD) on METH-induced cognitive impairment in a Novel Object Recognition task. A and B) Exploratory preference in training and retention sessions ofchronic vehicle- and METH-treated mice, injected on withdrawal day 4 with vehicle or MOD 90 and 30 mg/kg, respectively; the dotted line at 50% indicates equal preference forboth familiar and novel object, indicative of visual memory impairment. MOD 90 mg/kg retention session: ANOVA e Bonferroni. MOD 30 mg/kg retention session: Kruskal WallisANOVA on ranks. C and D) Total exploration time in training and retention sessions of chronic vehicle- and METH-treated mice, injected on withdrawal day 4 with vehicle or MOD90 and 30 mg/kg, respectively. E and F) Locomotion in training and retention sessions of chronic vehicle- and METH-treated mice, injected onwithdrawal day 4 with vehicle or MOD90 and 30 mg/kg, respectively. Time spent exploring objects and locomotion was analyzed with Ethovision XT 7.0 (Leesburg, Virginia). ANOVA e Bonferroni. Values indicatemean � SEM (N ¼ 12e22), different letters: p < 0.05.

B. González et al. / Neuropharmacology xxx (2014) 1e10 5

activity. For the 30 mg/kg modafinil group, two way ANOVAshowed significant treatment effect [F(3,33) ¼ 56.45, p < 0.001],but no novelty or interactive effects. For the 90 mg/kg modafinilgroup, two way ANOVA showed significant treatment[F(3,62) ¼ 53.30, p < 0.001], novelty [F(1,62) ¼ 10.43, p < 0.01], andinteractive [F(3,62) ¼ 3.33, p < 0.05] effects. Bonferroni post hoctests demonstrated that Vehicle and METH groups showed neithertreatment nor novelty effects on locomotion (Fig. 4A and B). Micegiven acute 30 mg/kg modafinil displayed only a main treatmenteffect, evidenced by MOD and METH-MOD groups showingincreased locomotion compared with Vehicle and METH groups

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(Fig. 4A). In agreement with locomotor responses obtained in micewhile performing the NOR task (Fig. 3E), the MOD and METH-MODgroups receiving 30 mg/kg modafinil showed similar locomotoreffects. In mice receiving acute 90 mg/kg MOD (Fig. 4B), we foundelevated locomotion in theMOD andMETH-MOD groups comparedto Vehicle and METH groups; also, with the 90 mg/kg dose theMETH-MOD group showed higher locomotion than the MODgroup. This is in agreement with locomotor responses obtained forthe same dose in the NOR task (Fig. 3F). We also found within theMETH-MOD group increased locomotion in the novelty groupwhen compared to the control group (p < 0.05).

methamphetamine-induced object recognition deficits and restores://dx.doi.org/10.1016/j.neuropharm.2014.02.002

Fig. 4. Effect of modafinil (MOD) on METH-induced changes in locomotion afternovelty exposure. Onwithdrawal day 4 after chronic vehicle or METH treatment (1 mg/kg, sc, once a day for 7 days) mice received acute modafinil and 1 h later performed a10 min Novelty session in which they were allowed to explore two equal novel objectsand euthanized immediately. Control mice were placed in the arena in the absence ofobjects and the same procedure was performed. A) Modafinil 30 mg/kg, B) Modafinil90 mg/kg. Locomotion was analyzed with Ethovision XT 7.0 (Leesburg, Virginia). Twoway ANOVA e Bonferroni. Values indicate the mean � SEM (N¼ 4e9), different letters:p < 0.05, *: different from METH-MOD control.

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3.2.2. Effect of acute modafinil on mice exposed to novel objectsduring methamphetamine withdrawal: ERK phosphorylation inmPFC

It was previously shown that one of the molecular mechanismsinvolved in METH-induced object recognition deficits involve afailure in mPFC ERK phosphorylation after exposure to novel ob-jects (Kamei et al., 2006). Thus, we decided to investigate if mod-afinil would be able to restore ERK signaling pathway in the mPFCafter a Novelty exposure. For both 30 mg/kg and 90 mg/kg mod-afinil experiments, two way ANOVA showed significant noveltyeffect [modafinil 30 mg/kg: F(1,35) ¼ 5.41, p < 0.05; modafinil90 mg/kg: F(1,47)¼ 10.33, p< 0.01], but no treatment or interactiveeffects. For both experiments, planned contrasts comparing controlvs. novelty within each treatment revealed a significant increase inERK phosphorylation in the mPFC of the Vehicle and MOD groupsimmediately after the Novelty session (p < 0.05), which was abol-ished in the METH group. We found that, in agreement with theretention results obtained in the NOR task, METH-MOD groupreceiving acute 30 mg/kg modafinil showed no novelty-inducedincrease in ERK phosphorylation (Fig. 5A). In mice that received90 mg/kg modafinil we observed that the novelty-induced ERKphosphorylationwas restored (p< 0.05) (Fig. 5B). The levels of total

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ERK did not differ between experimental groups in any case[modafinil 30 mg/kg: F(7,35) ¼ 1.75, p > 0.05; modafinil 90 mg/kg:F(7,47) ¼ 1.00, p > 0.05].

3.2.3. Effect of acute modafinil on mice exposed to novel objectsduring methamphetamine withdrawal: ERK phosphorylation in theNAc and hippocampus

The hippocampus participates in the formation and consolida-tion of newmemories and it was previously shown that MAPK andERK play important roles (Cammarota et al., 2008). Therefore, wemeasured novelty-induced ERK phosphorylation in the hippo-campus of METH-treated mice, with or without an injection of90 mg/kg modafinil (Fig. 6A). We found no changes in ERK phos-phorylation in the hippocampus in any group subjected to thenovelty effect [F(7,31) ¼ 1.00, p > 0.05] (Fig. 6A). The levels of totalERK did not differ between experimental groups in any condition[F(7,31) ¼ 1.24, p > 0.05].

There is evidence suggesting that the NAc is involved in thebehavioral response to novelty and other tasks that rely on famil-iarity discrimination (Sargolini et al., 2003; Nelson et al., 2010).Therefore, we evaluated novelty-induced ERK phosphorylation inthe NAc of METH-treated mice, with or without an injection of90 mg/kg modafinil (Fig. 6B). We found no changes in ERK phos-phorylation in NAc in any group subjected to the novelty effect[F(7,26) ¼ 0.35, p > 0.05]. The levels of total ERK did not differbetween experimental groups in any condition [F(7,26) ¼ 0.96,p > 0.05].

4. Discussion

The main finding of this study is that acute modafinil adminis-tration improved METH-induced object recognition memory defi-cits. METH-treated mice also showed a failure in ERKphosphorylation in the mPFC, which, in rodents, is detected shortlyafter a novel object is presented for exploration (Kamei et al., 2006;Nagai et al., 2007). Modafinil administration also restored novelty-induced ERK phosphorylation in the mPFC. To our knowledge, thisis the first study addressing modafinil’s beneficial effects on thevisual recognition deficits induced by METH in an animal model.Modafinil also showed positive effects in animal models of neuro-degeneration (Garcia et al., 2013). Interestingly, modafinil did notmodify cognition in naïve rats but was able to reverse memorydeficits induced by iron overload, a model of memory impairmentrelated to neurodegenerative disorders (Garcia et al., 2013).Furthermore, these authors used several doses of modafinil (0.75,7.5 or 75 mg/kg) and found that only the highest dose tested(75 mg/kg) was able to restore object recognition deficits (Garciaet al., 2013). These data are in agreement with results from thepresent study since we have observed that modafinil was onlyeffective at a dose of 90 mg/kg, while 30 mg/kg modafinil did notshow protective effects. Our results are also in agreement withprevious studies in humans, where modafinil administration wasable to improve cognitive deficits and prefrontal cortical functiononly in METH-dependent subjects (Ghahremani et al., 2011).Additionally, it has been shown that modafinil is more effective inindividuals who perform poorly on cognitive tasks relative to thosesubjects who perform better, indicating that modafinil does notenhance baseline performance but improves cognition in subjectswith evident cognitive impairments (Kalechstein et al., 2010; Finkeet al., 2010). Recent clinical studies showed that modafinil oraladministration in METH users with 7 days of abstinence, at a doserecommended for the treatment of sleeping disorders (200 mg,once daily), although improved several domains of executivefunction, had no effect on visual memory performance (Hesteret al., 2010). Accordingly, Finke et al., 2010 showed improved

methamphetamine-induced object recognition deficits and restores://dx.doi.org/10.1016/j.neuropharm.2014.02.002

Fig. 5. Effect of modafinil (MOD) on METH-induced changes in ERK phosphorylation inthe mPFC after novelty exposure. On withdrawal day 4 after chronic vehicle or METHtreatment (1 mg/kg, sc, once a day for 7 days) mice received acute modafinil and 1 hlater performed a 10 min novelty session, then euthanized immediately (noveltygroup). Control mice were placed in the arena in the absence of objects and the sameprocedure was performed (control group). A) Modafinil 30 mg/kg. B) Modafinil 90 mg/kg. Two way ANOVA followed by planned contrasts between control and noveltywithin each treatment. Values indicate the mean � SEM (N ¼ 4e9), *: significantlydifferent from control of the respective treatment.

Fig. 6. Effect of modafinil (MOD) on METH-induced changes in ERK phosphorylation inthe hippocampus and nucleus accumbens (NAc) after novelty exposure. On withdrawalday 4 after chronic vehicle or METH treatment (1 mg/kg, sc, once a day for 7 days) micereceived acute modafinil 90 mg/kg and 1 h later performed a 10 min novelty session,then were euthanized immediately (novelty group). Control mice were placed in thearena in the absence of objects and the same procedure was performed (controlgroup). A) Hippocampus, B) Nucleus accumbens (NAc). Two way ANOVA. Valuesindicate the mean � SEM (N ¼ 3e5).

B. González et al. / Neuropharmacology xxx (2014) 1e10 7

visual processing in participants with low performance baselinewhen using a higher modafinil dose (400 mg once daily). Alltogether, these results from clinical and pre-clinical studies suggestthat a high dose of modafinil might be needed in order to achieveimprovements in cognitive functions.

The mPFC mediates executive function and decision makingprocesses, and is therefore a key neuroanatomical region in

Please cite this article in press as: González, B., et al., Modafinil improvesprefrontal cortex ERK signaling in mice, Neuropharmacology (2014), http

addictive behaviors (Schoenbaum and Shaham, 2008). Studies us-ing functional magnetic resonance imaging have shown that PFCdysfunction in METH abusers is associated with cognitive impair-ment (Paulus et al., 2002), probably linked to deficits in DAneurotransmission (Tritsch and Sabatini, 2012). Although the ef-fects of modafinil on midbrain DA neuronal activity remaininconsistent, modafinil acts as a weak DAT inhibitor (Mereu et al.,2013). Consequently, it has been shown that acute modafiniladministration increased extracellular DA levels in the mPFC (de

methamphetamine-induced object recognition deficits and restores://dx.doi.org/10.1016/j.neuropharm.2014.02.002

B. González et al. / Neuropharmacology xxx (2014) 1e108

Saint Hilaire et al., 2001), the striatum of rodents (Dopheide et al.,2007; Nguyen et al., 2011; Young et al., 2011) and humans(Volkow et al., 2009). Acute modafinil also induced a strong acti-vation in those areas, measured using PET and confirmed by c-Fosimmunoreactivity (Gozzi et al., 2012). Modafinil was also found torescue visual object recognition in a rat model of schizophrenia(Redrobe et al., 2010), which is a condition that presents withdiminished dopamine neurotransmission in PFC (Okubo et al.,1997). Thus, modafinil’s ability to enhance cognitive performancein METH abusers might be associated with modafinil’s actions onthe DAT (Mereu et al., 2013), stimulating dopaminergic neuro-transmission in the mesocorticolimbic system.

It is known that ERK, a member of the MAPK family, is criticallylinked to dopaminergic neurotransmission and long-term memoryretention, but not memory acquisition or short term memory(Valjent et al., 2000; Nagai et al., 2007). ERK signaling is activatedby stimuli associated with synaptic activity and plasticity, like cal-cium influx and neurotrophins, and its activation mediates, in part,newmRNA and protein synthesis necessary for long-term memorystorage (Cammarota et al., 2008; Kelly et al., 2003). There is alsoaccumulating evidence implicating the ERK pathway in behavioralresponses to addictive drugs such as METH and cocaine (Mizoguchiet al., 2004; Valjent et al., 2000). ERK expression is especiallyabundant in the mesocorticolimbic DA system, a distribution thatunderscores the importance of the ERK signaling cascade inmediating DA function (Valjent et al., 2004). ERK signaling is alsoknown to be critically linked to DA D1 receptor activation, whichcouples with Gs proteins leading to cAMP and PKA cascade acti-vation (Valjent et al., 2000; Nagai et al., 2007). Activated ERK canpromote the expression of genes associated with memory andlearning, in part, by activating transcription factors such as CREBand Elk-1, which in turn activate gene transcription involved insynaptic and neuronal plasticity such as c-Fos, Zif268, etc.(Cammarota et al., 2008; Zhai et al., 2008). Our results indicate thatmodafinil modulates ERK phosphorylation in mPFC in a dose-treshold manner, which might be related to modafinil’s ability toenhance DA neurotransmission and stimulation of D1 receptors(Young and Geyer, 2010).

It is well known that repeated exposure to drugs of abuse resultsin a progressive and long-lasting enhancement of the locomotorresponse, a phenomenon termed psychomotor (or locomotor)sensitization (Robinson and Berridge, 1993). It is also of interest todiscuss our results showing that modafinil induced a cross-sensitization effect with METH. We showed that the same mod-afinil dose that enhanced visual retention in METH-treated mice,90 mg/kg, also elicited a cross-sensitization effect in locomotion. Aprevious study in mice also found locomotor cross-sensitizationbetween METH and modafinil assessed by challenging modafinil-pretreated mice with 1 mg/kg METH, and METH-pretreated micewith 50 mg/kg modafinil (Soeiro Ada et al., 2012). Our results oncross-sensitization between METH and modafinil raise the possi-bility that neuroadaptations induced by METH and evidenced byacute administration of modafinil might be related to the beneficialeffects of modafinil on cognition.

There is some discrepancy in the role of novelty-mediated ERKsignaling in the hippocampus, since ERK hyperphosphorylationafter training session was observed in rats (Kelly et al., 2003), butnot in mice (Kamei et al., 2006). In the present study, we found nonovelty-mediated changes in ERK phosphorylation in the hippo-campus of the experimental groups, in agreement with the studiesperformed by Kamei et al., 2006 in ICRmice. A possible explanationfor the differences observed between species may be that the studyperformed in rats analyzed specifically the dentate gyrus and CA1regions of the hippocampus, while the studies in mice where per-formed on the whole structure, which may have diluted the

Please cite this article in press as: González, B., et al., Modafinil improvesprefrontal cortex ERK signaling in mice, Neuropharmacology (2014), http

differences. Nonetheless, our results are in agreement with recentreports showing that the hippocampus, together with the peri-rhinal and the prefrontal cortices, is involved in the object locationand object-in-place tasks, which have a spatial processingcomponent, but not in the novel object recognition task (Forwoodet al., 2005; Barker and Warburton, 2011). We also found nonovelty-mediated ERK phosphorylation in the NAc of the experi-mental groups. The role of NAc in novelty processing is contro-versial, with reports indicating a NAc role only in tasks that involvespatial processing (Mele et al., 2004; Nelson et al., 2010), and re-ports indicating that the NAc also participates in visual object (non-spatial) processing (Sargolini et al., 2003). Our results indicate that,whether or not the NAc is involved in novelty processing, the ERKpathway in this structure was not affected by either METH ormodafinil.

Imaging studies show that drug abusers have marked decreasesin DA receptors and DA release. This decrease in DA function isassociated with reduced activity in brain areas that mediateinhibitory control and executive function, such as the cingulategyrus and the dorsolateral PFC. Deficits in DA activity, linked withprefrontal and striatal deregulation, play a central role in the loss ofcontrol and compulsive drug intake behavior (Volkow et al., 2009).Although the mechanisms underlying modafinil-induced im-provements in prefrontal function and cognitive performance haveyet to be delineated, our results further support the notion thatmodafinil represents an effective therapeutic intervention aimed atrestoring brain dopaminergic tone, and therefore restoring intra-cellular pathways like the MAPK-ERK pathway in the PFC. More-over, results from the present study further support the hypothesisthat drugs that could modulate ERK pathways might in turn beuseful for the treatment of the cognitive consequences of drugabuse. Specifically, our results point to modafinil as a good candi-date for the treatment of METH dependence, considering modafiniltherapy as an agonist-like replacement therapy (Herin et al., 2010).

Acknowledgments

Dr. Bisagno has been authorized to study drug abuse substancesin animal models by A.N.M.A.T. (National Board of Medicine FoodandMedical Technology, Ministerio de Salud, Argentina). Dr. BetinaGonzalez is a recipient of a Postdoctoral Award from FundaciónBunge y Born. This work is supported by grants PIP11420100100072 and PICT 2012-0924, Argentina, and by NIH awardP20 GM103425 to the Center for Translational Neuroscience.

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