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Behavioural Pharmacology

The selective norepinephrine reuptake inhibitor atomoxetine counteracts behavioralimpairments in trimethyltin-intoxicated rats

Alessandra Tamburella, Vincenzo Micale, Carmen Mazzola, Salvatore Salomone, Filippo Drago ⁎Department of Clinical and Molecular Biomedicine, Section of Pharmacology and Biochemistry, Catania University, Catania, Italy

a b s t r a c ta r t i c l e i n f o

Article history:Received 23 January 2012Received in revised form 17 February 2012Accepted 26 February 2012Available online 8 March 2012

Keywords:Attention deficit/hyperactivity disorderSpontaneously hypertensive ratTrimethyltinAtomoxetineMethylphenidateCognition

This study was carried out to assess the behavioral effects of the non-psychostimulant drug atomoxetine, inrats prenatally-exposed to the organic compound trimethyltin chloride (TMT) and in spontaneously hyper-tensive rat (SHR), two rodent models of Attention Deficit/Hyperactivity Disorder (ADHD). At birth, neonatalreflexes (righting, cliff aversion, forelimb placing, forelimb grasping, bar holding and startle) had an earlieronset (i.e. percent of appearance) and completion (maximum appearance, i.e. 100% of the brood exhibitingeach reflex) in prenatally TMT-exposed and SHR pups as compared to control groups. Two months afterbirth, TMT-exposed and SHR rats showed impaired cognitive performances in both the step-through passiveavoidance test and the shuttle box active avoidance test. Atomoxetine (1, 3 and 6 mg/kg, i.p.), already at thelowest dose tested, improved learning and memory capacity of prenatally TMT-exposed rats and SHR; whilemethylphenidate (1, 3 and 6 mg/kg, i.p.), used here as positive control, elicited a significant cognitive enhanc-ing effect only at the higher doses. In the open field test, both TMT-exposed rats and SHR displayed enhancedlocomotor activity. Methylphenidate further increased locomotor activity in all groups, whereas atomoxetinereduced the enhanced locomotor activity of TMT-exposed rats and SHR down to the level of controls. Theseresults suggest that prenatal TMT-exposure could be considered as a putative experimental model of ADHDand further support the effectiveness of atomoxetine in the ADHD pharmacotherapy. Furthermore, despitethe similar effect of the two drugs on cognitive tasks, they exhibit distinct profiles of activity on locomotion,in ADHD models.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The Attention-Deficit/Hyperactivity Disorder (ADHD) is a com-mon neurobehavioral disorder, characterized by hyperactivity, impul-sivity, deficit in attention and cognition, affecting as much as 5–10% ofschool aged children in United States (American Academy ofPediatrics, 2000). Although the biological bases of ADHD remain part-ly unknown, increasing evidence suggests that ADHD is associatedwith a dysfunction of catecholaminergic (i.e. dopamine and norepi-nephrine) systems (Biederman, 2005). The first-line medicationtreatment to control the behavioral symptoms of ADHD is repre-sented by the psychostimulant methylphenidate, that potentiatesthe catecholaminergic neurotransmission, but its usefulness is limit-ed, because many patients do not respond to psychostimulant thera-py. Moreover, methylphenidate is a controlled substance, associatedto drug abuse and diversion (Holman, 1994). A substantial progressin the pharmacological treatment for ADHD has recently beenachieved by approval of a non-psychostimulant agent, such as

atomoxetine. This drug selectively inhibits norepinephrine reuptakeand enhances catecholaminergic transmission in the prefrontal cor-tex, but does not act on the nucleus accumbens, a brain area associat-ed with rewarding behaviors and addiction. Hence, atomoxetine doesnot show the drug abuse potential typical of psychostimulants(Garnock-Jones and Keating, 2009; Kollins et al., 2009). The organotintrimethyltin chloride (TMT) produces a selective central nervous sys-tem (CNS) damage (i.e. hippocampal neuronal degeneration) in prena-tally exposed rodents associated to cognitive impairment, hyperactivityand aggressiveness, similar to those found in other, well validated,ADHD animal models (Russell et al., 2005). To date, however, thereare no data concerning the effects of the twomost commonADHDmed-ications (i.e. atomoxetine and methylphenidate) on the behavioralchanges induced by TMT intoxication.

Based on these premises, this study was undertaken to assess thephenotype (i.e. motor and cognitive performance) of prenatally TMT-intoxicated rats, as compared to spontaneously hypertensive rats(SHR), that show the full range of ADHD-like symptoms (Sagvolden,2000). The expression of neonatal reflexes was evaluated as anindex of brain maturation (Fox, 1965). Furthermore, the effects ofacute treatment with atomoxetine on both cognitive and locomotoractivity were assessed in TMT prenatally-exposed rats and in SHR,by testing in the passive or active avoidance tests and in the open

European Journal of Pharmacology 683 (2012) 148–154

⁎ Corresponding author at: Department of Clinical andMolecular Biomedicine, Sectionof Pharmacology and Biochemistry, Catania University, Viale A. Doria 6, 95125, Catania,Italy. Tel.: +39 095 7384236; fax: +39 095 7384238.

E-mail address: fdrago@tin.it (F. Drago).

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field, respectively. Comparative data for the psychostimulant methyl-phenidate, under the same experimental conditions, were obtained.

2. Material and methods

2.1. Animals

Pregnant Wistar, Wistar Kyoto (WKY) and spontaneously hyper-tensive rats (SHR), at gestational day 10, were purchased fromCharles River (Calco, Italy). Wistar rats were used for the TMT-model (where the control was represented by TMT-unexposed Wis-tar rats). WKY rats were used as control for the SHR genetic model,because different rat strains comprise different gene pools thatcould lead to differences in susceptibility to pharmacological treat-ment (Fox et al., 2002; Segal and Kuczenski, 1987); WKY have a ge-netic background closely related to SHR, having been originally usedto develop SHR by inbreeding (Okamoto and Aoki, 1963). For atleast 1 week prior to the experiments, the animals were housed insingle cages at a temperature of 22±1 °C and under a 12-h light/dark cycle (lights on between 8:00 and 20:00 h), with food and tapwater available ad libitum. At birth, the number of newborn pupswas counted and the presence of malformations was recorded. Malerats were separated at weaning and further used for the behavioralexperiments, two months after birth. Animals were randomlyassigned to treatment groups and tested only once in the behavioralexperiments. All experiments were carried out according to the Euro-pean Community Council 86/609/EEC and efforts were made to min-imize animal suffering and to reduce the number of animals used. Therationale, design and methods of this study have been approved bythe Ethical Committee for Animal Research, University of Catania.

2.2. Drugs and experimental design

All compounds were administered in a volume of 1 ml/kg bodyweight. Trimethyltin (TMT, 9 mg/kg; Sigma, St. Louis, MO, USA) wasdissolved in saline and injected intraperitoneally (i.p.) to Wistar ratsat gestational day 15, considered crucial for brain development inrats (Granados et al., 1995; Zoli et al., 1990). Atomoxetine hydrochlo-ride (1, 3 or 6 mg/kg; Eli-Lilly, Indianapolis, IN, USA) and methylphe-nidate hydrochloride (1, 3 or 6 mg/kg; Sigma), were freshlysolubilized in saline and administered i.p. to prenatally TMT-exposed rats or to SHR (2-month old), 1 h prior to the behavioraltests. The doses of the compounds were selected based on previousstudies (Fox et al., 2002; Tzavara et al., 2006). Control groups weresubjected to the same procedures and injected with vehicle. Four dif-ferent experiments with 10 animals per group were carried out, asdescribed below [overall 28 groups, resulting from two differentADHD models with their respective controls (i.e. 4 groups: VHC,TMT, WKY, SHR), each group subjected to 7 different acute treat-ments (i.e. vehicle, methylphenidate 1, 3 and 6 mg/kg, atomoxetine1, 3 and 6 mg/kg)].

2.3. Behavioral experiments

2.3.1. Experiment 1 — examination of neurobehavioral developmentDevelopment of neonatal behavior was studied by applying a bat-

tery of tests, as previously described (Drago et al., 1999; Lo Pumo etal., 2006; Nicosia et al., 2003). Starting on postnatal day 1, newbornpups were daily weighed and observed for neonatal reflexes, until“maximum appearance” was scored (i.e., 100% of the brood wasfound to exhibit the full repertoire of reflexes). The following reflexeswere scored: cliff aversion (the rat withdraws from the edge of a flatsurface when its snout and forepaws are placed over a cliff 60-cmhigh), righting (the rat is capable of rapidly returning to its feetwhen placed on its back), forelimb placing (the rat places its forepawup to a cardboard when it has been stroked against the dorsal surface

of the paw), forelimb grasping (the rat grasps strongly the barrel ofthe 16-gauge needle, 1.0-mm diameter, when it is touched againstthe palm of each forepaw), bar holding (the rat holds itself on to awooden stick, 2.0-mm diameter, for 5 s) and startle (the rat shows awhole body startle response when a loud snap of the fingers is pro-duced at 10-cm distance).

2.3.2. Experiment 2 — testing passive avoidance behaviorRetention of passive avoidance reaction was evaluated in a step-

through type of passive avoidance test, as previously described(Drago et al., 1999; Lo Pumo et al., 2006). The apparatus for thestep-through passive avoidance test was an automated shuttle box(Ugo Basile, Comerio, Italy), divided into an illuminated compart-ment and a dark compartment of the same size by a wall with a guil-lotine door. Adaptation training was followed by a single trial inwhich the animals were placed individually into the illuminatedcompartment and allowed to enter the dark box. Three further trialswere carried out on the next day, with an inter-trial interval of5 min. After the third trial, the animals received a single 2-s unavoid-able scrambled foot shock (0.25 mA) immediately after entering thedark compartment. The retention of passive avoidance response wasmeasured 1 and 7 days after the learning trial. Each animal was againput into the illuminated compartment and the latency to reenter thedark compartment was recorded. No foot shock was delivered whilethe retention test was performed. The maximum cut-off time forstep-through latency was 300 s.

2.3.3. Experiment 3 — testing active avoidance behaviorShuttle box active avoidance acquisition was evaluated in a single

session, as described elsewhere (Lo Pumo et al., 2006; Micale et al.,2006). Briefly, the rat was trained to avoid the unconditioned stimu-lus (US) of a scrambled electrical foot shock (0.20 mA) deliveredthrough the grid floor. The conditioned stimulus (CS) was a buzzerpresented for 5 s prior to the US. If no escape occurred within 20 sof CS/US presentation, the shock was terminated. A maximum of 30conditioning trials were given with a variable inter-trial interval aver-aging 60 s. The learning criterion was five consecutive conditionedavoidance responses. For those animals that reached the criterion inless than 30 trials, the remaining trials until 30 were considered asconditioned avoidance responses.

2.3.4. Experiment 4 — testing motor activityRats were studied for spontaneous motor activity as previously

described (Tamburella et al., 2009). The experiment was performedin a soundproof and moderately illuminated (~50 lx) cubic observa-tion chamber (2×2×2 m) between 10:00 and 17:00 h, using awhite wooden open field (100×100 cm, walls 40-cm high). At the be-ginning of the test, animals were placed gently in the center of thearena and allowed to explore. The horizontal activity of each animal(ambulatory) was defined as the number of squares crossed with atleast the forelegs, while the vertical activity (rearing) was definedas the number of times the rat stood upright on its hind limbs.Motor activity was assessed in 5-min sessions, recorded on a tapeusing a video camera (Hitachi Videocam) and scored by a video track-ing software (Ugo Basile).

2.4. Statistical analysis

Data were analyzed by two-way analysis of variance (ANOVA) andthe post hoc Dunnett's test for multiple comparisons. The Fisher'sexact t-test was used for frequencies (comparisons of percent dataof reflex appearance and comparisons of percent data of learners). AP value of 0.05 or less was considered as indicative of a significantdifference.

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3. Results

3.1. Neurological reflexes

The appearance rate of neonatal reflexes in prenatally TMT-exposed rats and in SHR is shown in Fig. 1. As similar results wereobtained from Wistar rats prenatally exposed to vehicle and WKYrats, data from these two control groups were pooled. At birth, no dif-ference was found in total number of pups per litter, body weight,malformations, eye opening time (data not shown). The percent ap-pearance (i.e., cumulative number of pups exhibiting reflexes overthe time) and the completion (maximum appearance, i.e. 100% ofpups exhibiting each reflex) of neonatal reflexes had an earlieronset both in prenatally TMT-exposed rats and in SHR as comparedto controls. Statistical analysis revealed several time points in whichthe percentage of pups exhibiting righting (A), cliff aversion (B), fore-limb placing (C), forelimb grasping (D), bar holding (E) and startle (F)was significantly higher in TMT-exposed and in SHR as compared tocontrols (Pb0.05; Pb0.01; Pb0.001). Furthermore, no differencewas found in the appearance of neonatal reflexes between prenatallyTMT-exposed rats and SHR.

3.2. Passive avoidance test

The effects of treatment with atomoxetine or methylphenidate onbehavioral responses of TMT-exposed rats and SHR in the passiveavoidance task are shown in Fig. 2. In the TMT model (Fig. 2, A, B),two-way ANOVA (factor 1: TMT; factor 2: treatment) revealed a signif-icant effect of TMT [1st retention test (F1,126=46.22, Pb0.001), 2ndretention test (F1,126=31.13, Pb0.001)], treatment [1st retentiontest (F6,126=24.22, Pb0.001); 2nd retention test (F6,126=15.13,Pb0.001) and a TMT×treatment interaction [1st retention test (F6,126=14.14, Pb0.001); 2nd retention test (F6,126=10.12, Pb0.001)]. Posthoc analysis revealed that TMT-exposed vehicle treated rats showedan impaired retention capacity as compared to the respective control

group (prenatally vehicle-exposed, vehicle-injected rats) in bothretention tests (Pb0.001). Methylphenidate, at doses of 3 and6 mg/kg, improved the cognitive performance of TMT-exposedrats (Pb0.01) in the 1st retention test, but only at the dose of6 mg/kg elicited a significant effect in the 2nd retention test(Pb0.05). Atomoxetine significantly increased the latency time inboth retention tests already at the lowest tested dose (1 mg/kg,Pb0.001); at the highest dose tested (6 mg/kg), there was a ten-dency toward a reduction of atomoxetine effect on the passiveavoidance responses of TMT-exposed animals.

In the SHR model (Fig. 2, C, D), two-way ANOVA (factor 1: strain;factor 2: treatment) revealed a significant effect of strain [1st retentiontest (F1,126=73.42, Pb0.001); 2nd retention test (F1,126=65.31,Pb0.001)], treatment [1st retention test (F6,126=17.11, Pb0.01);2nd retention test (F6,126=10.03, Pb0.05)] and a strain×treatmentinteraction [1st retention test (F6,126=13.10, Pb0.01); 2nd retentiontest (F6,126=8.07, Pb0.05)]. Post hoc analysis revealed that SHRvehicle-treated rats showed a reduced latency to re-enter the darkbox in both retention tests, an index of impaired memory capacity, ascompared to the control group (WKY vehicle-treated rats, Pb0.001).Methylphenidate (3 or 6 mg/kg) improved the memory capacity ofSHR group in the 1st, but not in the 2nd retention test (Pb0.05), whileatomoxetine (1 or 3 mg/kg) elicited significant effects in both retentiontests (Pb0.05, Pb0.01). At the highest dose tested (6 mg/kg), however,the effect of atomoxetine on the passive avoidance responses of SHRde-clined down to the level of vehicle-treated animals.

3.3. Active avoidance test

The effects of treatment with atomoxetine or methylphenidate onbehavioral responses of TMT-exposed rats and SHR in the activeavoidance task are shown in Fig. 3. In the TMT model (Fig. 3, A, B),two-way ANOVA revealed a significant effect of TMT (F1,126=35.42, Pb0.001), treatment (F6,126=25.12, Pb0.001) and a TMT×treatment interaction (F6,126=13.12, Pb0.01). Similarly, in the

Fig. 1. Appearance rate of righting (A), cliff aversion (B), forelimb placing (C), forelimb grasping (D), bar holding (E) and startle reflexes (F), in prenatally trimethyltin-exposed rats(TMT) and in spontaneously hypertensive rats (SHR). The appearance of each reflex was scored each day, beginning on day 1 after birth, according to the method described in thetext. TMT (9 mg/kg) or vehicle was administered i.p. to pregnant Wistar mothers at gestational day 15. Two control groups were used (Wistar rats prenatally exposed to vehicle forTMT and Wistar Kyoto rats for SHR); however, because data from these two control groups did not differ, they were pooled (Control). Values are mean of percent cumulative ap-pearance of each reflex (episode) in each day, per group of animals (n=10).*Pb0.05, **Pb0.01, ***Pb0.001 vs control. Fisher's exact t-test.

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SHR model (Fig. 3, C, D) there was a significant effect of strain(F1,126=43.24, Pb0.001), treatment (F6,126=23.02, Pb0.001) anda strain×treatment interaction (F6,126=11.09, Pb0.05). Post hocanalysis revealed that both TMT-exposed and SHR rats exhibited a re-duction in number of conditioned avoidance responses (Pb0.001),that was partially reversed by methylphenidate or atomoxetine(Pb0.05; Pb0.01). Both TMT and SHR also exhibited a more pro-nounced reduction in percent of learners, that was reversed by phar-macological treatments (Pb0.001).

3.4. Locomotor activity

The effects of treatment with atomoxetine or methylphenidateon locomotor activity of TMT-exposed rats and SHR are shownin Fig. 4. In the TMT model (Fig. 4, A, B), two-way ANOVA revealeda significant effect of TMT (crossing: F1,126=12.32, Pb0.01; rearing:F1,126=10.21, Pb0.05) and treatment (crossing: F6,126=15.03,pb0.01; rearing: F6,126=11.03, Pb0.05), but not a TMT×treatmentinteraction, indicating that drug-treatment affected locomotor activi-ty independently of prenatal exposure to TMT. Similarly, in the SHRmodel (Fig. 4, C, D), two-way ANOVA revealed a significant effect ofstrain (crossing: F1,126=9.14, Pb0.05; rearing: F1,126=11.11,Pb0.05) and treatment (crossing: F6,126=7.51, Pb0.05; rearing:F6,126=13.05, Pb0.05), but not a strain×treatment interaction, in-dicating that drug-treatment affected locomotor activity indepen-dently of strain.

Post hoc analysis showed that TMT and SHR displayed an in-creased locomotor activity as compared to controls (prenatallyvehicle-exposed, vehicle-injected rats and WKY vehicle- injectedrats, Pb0.01). Methylphenidate (3 or 6 mg/kg) increased locomotoractivity in all groups (Pb0.05; Pb0.01); atomoxetine at all doses, incontrast, did not modify locomotor activity in VHC and WKY, but sig-nificantly reduced locomotor activity in TMT and SHR down to thelevel of controls (Pb0.05; Pb0.01).

4. Discussion

The present results show, for the first time, that prenatally TMT-exposed rats have a behavioral phenotype similar to that of SHR, awidely accepted rodent model of ADHD (Sagvolden, 2000). First, byanalyzing neonatal behavior, we found that the appearance and com-pletion of neonatal reflexes had an earlier onset and an earlier com-pletion in prenatally TMT-exposed and in SHR pups as compared tocontrols. Worthy of note, WKY rats, the control in the SHR model,showed the same phenotype of Wistar strain, that was used in theTMT model, i.e. the development of reflexes was similar in Wistarrats not exposed to TMT and in WKY, suggesting that the appearanceof neonatal reflexes in our study was not strain-specific. Neonatal re-flexes are considered an index of brain maturation and changes intheir development and expression could represent a predictive factorfor other behavioral modifications in adulthood (Fox, 1965; Iezhitsaet al., 2001). Consistent with this view, prenatally TMT-exposed ratsand SHR showed impaired cognitive performance when tested in cog-nitive tasks. We did not directly examine the mechanisms underlyingthe effect, in adulthood, of early TMT-exposure; however, it is wellrecognized that TMT induces neuronal damage in hippocampus ofrodents and humans and affects neurotransmitter systems, such ascholinergic, GABAergic and glutamatergic (Cannon et al., 1994;Earley et al., 1992; Naalsund et al., 1985; Patel et al., 1990; Wilsonet al., 1986), which, in turn, could affect the behavioral performance(Kim et al., 2007; Paule et al., 1986; Woodruff et al., 1991). Further-more, activation of caspase 3 has been observed following TMT-exposure, whichmay contribute to apoptotic and neurodegenerativeprocesses in brain of rats prenatally exposed to TMT (Geloso et al.,2002; Ishida et al., 1997; Jenkins and Barone, 2004; Kawada et al.,2008).

Among the diverse genetic and drug-induced animal models ofADHD, SHR has often been considered the most validated one, becauseit “naturally” displays ADHD symptoms, including hyperactivity,

Fig. 2. Effects of drug-treatment on passive avoidance response in prenatally trimethyltin-exposed rats (TMT), in spontaneously hypertensive rats (SHR) and in their controls (pre-natally vehicle-exposed, VHC and Wistar Kyoto, WKY, respectively). The assessment of retention capacity was measured 1 and 7 days after the learning trial. Methylphenidate(MPH; 1, 3 or 6 mg/kg), atomoxetine (ATX; 1, 3 or 6 mg/kg) or vehicle (VHC) were injected i.p. to TMT and VHC (A, B) or SHR and WKY (C, D), 1 h prior to the 1st retentiontest. Values are means±standard error of mean (vertical bars) of latency time to re-enter the dark box, expressed in s (n=10 rats per group).*Pb0.05, **Pb0.01, ***Pb0.001 vsTMT VHC or SHR VHC; †††Pb0.001 vs control (VHC, VHC-injected); ‡‡‡Pb0.001 vs control (WKY, VHC-injected). Dunnett's test for multiple comparisons.

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Fig. 4. Effects of drug-treatment on locomotor activity in prenatally trimethyltin-exposed rats (TMT), in spontaneously hypertensive rats (SHR) and in their controls (prenatallyvehicle-exposed, VHC and Wistar Kyoto, WKY, respectively). Methylphenidate (MPH; 1, 3 or 6 mg/kg), atomoxetine (ATX; 1, 3 or 6 mg/kg) or vehicle (VHC) were injected i.p.to TMT and VHC (A, B) or SHR and WKY (C, D) 1 h prior to behavioral testing. Values are means±standard error of mean (vertical bars) of the total number of squares crossed(crossing) and rearing episodes, scored in a 5-min session (n=10 rats per group).**Pb0.01 vs control (VHC VHC-injected or WKY VHC-injected); †Pb0.05, ††Pb0.01 vs TMTVHC or SHR VHC. Dunnett's test for multiple comparisons.

Fig. 3. Effects of drug-treatment on active avoidance response in prenatally trimethyltin-exposed rats (TMT), in spontaneously hypertensive rats (SHR) and in their controls (pre-natally vehicle-exposed, VHC and Wistar Kyoto, WKY, respectively). Methylphenidate (MPH; 1, 3 or 6 mg/kg), atomoxetine (ATX; 1, 3 or 6 mg/kg) or vehicle (VHC) were injectedi.p. to TMT and VHC (A, B) or SHR andWKY (C, D) 1 h prior to behavioral testing. Values are means±standard error of mean (vertical bars) of total number of conditioned avoidanceresponses and percent of learners (n=10 rats per group).*Pb0.05, **Pb0.01, ***Pb0.001 vs TMT VHC or SHR VHC; †††Pb0.001 vs control (VHC, VHC-injected); ‡‡‡Pb0.001 vs con-trol (WKY, VHC-injected). Dunnett's test for multiple comparisons.

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impulsivity and cognitive deficits, whose mechanisms are object of in-tense research and still not fully understood. A dysfunctional calciumsignalingmechanism, that has been found in SHR,may affect the neuro-transmitter release and underlie the behavioral abnormalities of thisstrain (Pamplona et al., 2009; Pires et al., 2009; Prediger et al., 2005;Sagvolden, 2000). To validate the TMT model and its potential fordrug screening in ADHD, we evaluated here methylphenidate and ato-moxetine, two drugs widely prescribed for ADHD. We found that bothdrugs ameliorated the cognitive performance of prenatally TMT-exposed rats (this is, in fact, the first evidence of this type), as well asof SHR, when tested in two different cognitive tasks, the passive andthe active avoidance tests. In particular, atomoxetine improved the cog-nitive performance already at the lowest tested dose (1 mg/kg), result-ing more potent than methylphenidate on these learning measures.Atomoxetine is a highly specific norepinephrine reuptake inhibitorwith low affinity for other monoamine transporters and receptors(Bymaster et al., 2002), whereas methylphenidate, as other amphet-amines, induces redistribution of catecholamines from synaptic vesiclesto the cytosol, and induction of reverse transport through plasmamem-brane uptake carrier (Sulzer et al., 2005). Despite these different profilesof action, however, both compounds displayed the same activity in thepassive and active avoidance paradigms, in both the TMT and the SHRmodel, without affecting the controls (prenatally vehicle-exposed andWKY rats). This latter finding suggests that the effectiveness of methyl-phenidate and atomoxetine in enhancing cognition may be mainlyexerted in experimental pathologic conditions, such as after TMT expo-sure or in SHR strain, rather than in normal condition. The effectivenessof atomoxetine or methylphenidate to improve cognitive deficits inexperimental models of ADHD has been also shown by others(Sagvolden, 2000). Here we found that atomoxetine and methylphe-nidate were similarly effective in two models, involving 3 differentstrains (Wistar, WKY and SHR), suggesting that the susceptibilityto pharmacological treatment in our study was not strain-specific.When looking at the effects of drug-treatment on locomotor activitywe found important differences between methylphenidate and ato-moxetine. Methylphenidate increased locomotor activity in allgroups, whereas atomoxetine did not change locomotor activity incontrols but reduced it in TMT and SHR. Of note, both TMT and SHRdisplayed an increased locomotion compared to their respectivecontrols. The effect of acute administration of methylphenidate onlocomotor activity we observed here is consistent with what isknown since longtime (for review see Askenasy et al., 2007). In con-trast, little is known on the effects of atomoxetine on locomotor ac-tivity in rat models of ADHD (Viggiano et al. 2004); however,consistent with our observation, data obtained in mice show thatatomoxetine decreases locomotion (Tsuchida et al., 2009). It shouldbe emphasized here that our study has been carried out in acute con-ditions, i.e. following a single drug-injection. Conceivably, chronicadministration of methylphenidate and atomoxetine may have dif-ferent effects on locomotion; in this respect, data with methylpheni-date are controversial, showing both increase (Gaytan et al., 1997;Yang et al., 2006) and, more recently, decrease (Avital et al., 2011;Bethancourt et al., 2011); on the other hand, data with chronic ato-moxetine are not available. Future studies should therefore pay at-tention to the chronic effects of these drug-treatments, becauselong-term administration to animals would more closely mimic theclinical ADHD contest.

In conclusion, we propose that prenatally TMT-exposed rat couldrepresent an animal model of ADHD, because it mimics the same phe-notype of SHR, a well validated animal model of ADHD, both in termsof cognitive deficits and of hyperlocomotion. Furthermore, clinicallyused stimulants, such as methylphenidate, and non-stimulants suchas atomoxetine, counteract the effects of prenatal TMT-exposure,opening new perspectives on the patho-physiology of ADHD in ex-perimental models. Further studies are required to confirm the effec-tiveness of atomoxetine and methylphenidate in the TMT model,

following chronic treatment, and to characterize the effect of thesedrugs at the molecular level.

Acknowledgments

We are grateful to Eli-Lilly (Italy) for the generous gift of atomox-etine. These experiments were supported by the PhD InternationalSchool Program in Neuropharmacology, University of Catania MedicalSchool.

References

American Academy of Pediatrics, 2000. Clinical practice guideline: diagnosis and eval-uation of the child with attention-deficit/hyperactivity disorder. Pediatrics, 105.American Academy of Pediatrics, pp. 1158–1170.

Askenasy, E.P., Taber, K.H., Yang, P.B., Dafny, N., 2007. Methylphenidate (Ritalin):behavioral studies in the rat. Int. J. Neurosci. 117, 757–794.

Avital, A., Dolev, T., Aga-Mizrachi, S., Zubedat, S., 2011. Environmental enrichment pre-ceding early adulthood methylphenidate treatment leads to long term increase ofcorticosterone and testosterone in the rat. PLoS One 6, e22059.

Bethancourt, J.A., Vásquez, C.E., Britton, G.B., 2011. Sex-dependent effects of long-termoralmethylphenidate treatment on spontaneous and learned fear behaviors. Neurosci.Lett. 496, 30–34.

Biederman, J., 2005. Attention-deficit/hyperactivity disorder: a selective overview. Biol.Psychiatry 57, 1215–1220.

Bymaster, F.P., Katner, J.S., Nelson, D.L., Hemrick-Luecke, S.K., Threlkeld, P.G.,Heiligenstein, J.H., Morin, S.M., Gehlert, D.R., Perry, K.W., 2002. Atomoxetine in-creases extracellular levels of norepinephrine and dopamine in prefrontal cortexof rat: a potential mechanism for efficacy in attention deficit/hyperactivity disorder.Neuropsychopharmacology 27, 699–711.

Cannon, R.L., Hoover, D.B., Baisden, R.H., Woodruff, M.L., 1994. The effect of time fol-lowing exposure to trimethyltin (TMT) on cholinergic muscarinic receptor bindingin rat hippocampus. Mol. Chem. Neuropathol. 23, 47–62.

Drago, F., Di Leo, F., Giardina, L., 1999. Prenatal stress induces body weight deficit andbehavioural alterations in rats: the effect of diazepam. Eur. Neuropsychopharma-col. 9, 239–245.

Earley, B., Burke, M., Leonard, B.E., 1992. Behavioural, biochemical and histological ef-fects of trimethyltin (TMT) induced brain damage in the rat. Neurochem. Int. 21,351–366.

Fox, M.W., 1965. Reflex ontogeny and behavioral development of the mouse. Anim.Behav. 13, 234–245.

Fox, G.B., Pan, J.B., Esbenshade, T.A., Bennani, Y.L., Black, L.A., Faghih, R., Hancock,A.A., Decker, M.W., 2002. Effects of histamine H(3) receptor ligands GT-2331and ciproxifan in a repeated acquisition avoidance response in the spontaneouslyhypertensive rat pup. Behav. Brain Res. 131, 151–161.

Garnock-Jones, K.P., Keating, G.M., 2009. Atomoxetine: a review of its use in attention-deficit hyperactivity disorder in children and adolescents. Paediatr. Drugs 11,203–226.

Gaytan, O., Ghelani, D., Martin, S., Swann, A., Dafny, N., 1997. Methylphenidate: diurnaleffects on locomotor and stereotypic behavior in the rat. Brain Res. 777, 1–12.

Geloso, M.C., Vercelli, A., Corvino, V., Repici, M., Boca, M., Haglid, K., Zelano, G., Michetti,F., 2002. Cyclooxygenase-2 and caspase 3 expression in trimethyltin-induced apo-ptosis in the mouse hippocampus. Exp. Neurol. 175, 152–160.

Granados, L., Cintra, L., Aguilar, A., Corkidi, G., Kemper, T., Morgane, P., Díaz-Cintra, S.,1995. Mossy fibers of the hippocampal formation in prenatal malnourished rats.Bol. Estud. Med. Biol. 43, 3–11.

Holman, R.B., 1994. Biological effects of central nervous system stimulants. Addiction89, 1435–1441.

Iezhitsa, I.N., Spasov, A.A., Bugaeva, L.I., 2001. Effects of bromantan on offspring matu-ration and development of reflexes. Neurotoxicol. Teratol. 23, 213–222.

Ishida, N., Akaike, M., Tsutsumi, S., Kanai, H., Masui, A., Sadamatsu, M., Kuroda, Y.,Watanabe, Y., McEwen, B.S., Kato, N., 1997. Trimethyltin syndrome as hippocampalde generation model: temporal changes and neurochemical features of seizuresusceptibility and learning impairment. Neuroscience 81, 1183–1191.

Jenkins, S.M., Barone, S., 2004. The neurotoxicant trimethyltin induces apoptosis viacaspase activation, p38 protein kinase, and oxidative stress in PC12 cells. Toxicol.Lett. 147, 63–72.

Kawada, K., Yoneyama, M., Nagashima, R., Ogita, K., 2008. In vivo acute treatment withtrimethyltin chloride causes neuronal degeneration in the murine olfactory bulband anterior olfactory nucleus by different cascades in each region. J. Neurosci.Res. 86, 1635–1646.

Kim, M.J., Choi, S.J., Lim, S.T., Kim, H.K., Heo, H.J., Kim, E.K., Jun, W.J., Cho, H.Y., Kim, Y.J.,Shin, D.H., 2007. Ferulic acid supplementation prevents trimethyltin-induced cog-nitive deficits in mice. Biosci. Biotechnol. Biochem. 71, 1063–1068.

Kollins, S.H., English, J., Robinson, R., Hallyburton, M., Chrisman, A.K., 2009. Reinforcingand subjective effects of methylphenidate in adults with and without attentiondeficit hyperactivity disorder (ADHD). Psychopharmacology 204, 73–83.

Lo Pumo, R., Bellia, M., Nicosia, A., Micale, V., Drago, F., 2006. Long-lasting neurotoxicityof prenatal benzene acute exposure in rats. Toxicology 223, 227–234.

Micale, V., Leggio, G.M., Mazzola, C., Drago, F., 2006. Cognitive effects of SL65.0155, aserotonin 5-HT4 receptor partial agonist, in animal models of amnesia. Brain Res.1121, 207–215.

153A. Tamburella et al. / European Journal of Pharmacology 683 (2012) 148–154

Author's personal copy

Naalsund, L.U., Allen, C.N., Fonnum, F., 1985. Changes in neurobiological parameters inthe hippocampus after exposure to trimethyltin. Neurotoxicology 6, 145–158.

Nicosia, A., Giardina, L., Di Leo, F., Medico, M., Mazzola, C., Genazzani, A.A., Drago, F.,2003. Long-lasting behavioral changes induced by pre-or neonatal exposure todiazepam in rats. Eur. J. Pharmacol. 469, 103–109.

Okamoto, K., Aoki, K., 1963. Development of a strain of spontaneously hypertensiverats. Jpn. Circ. J. 27, 282–293.

Pamplona, F.A., Pandolfo, P., Savoldi, R., Prediger, R.D., Takahashi, R.N., 2009. Environ-mental enrichment improves cognitive deficits in Spontaneously HypertensiveRats (SHR): relevance for Attention Deficit/Hyperactivity Disorder (ADHD). Prog.Neuropsychopharmacol. Biol. Psychiatry 33, 1153–1160.

Patel, M., Ardelt, B.K., Yim, G.K., Isom, G.E., 1990. Interaction of trimethyltin with hippo-campal glutamate. Neurotoxicology 11, 601–608.

Paule, M.G., Reuhl, K., Chen, J.J., Ali, S.F., Slikker, W., 1986. Developmental toxicology oftrimethyltin in the rat. Toxicol. Appl. Pharmacol. 84, 412–417.

Pires, V.A., Pamplona, F.A., Pandolfo, P., Fernandes, D., Prediger, R.D., Takahashi, R.N.,2009. Adenosine receptor antagonists improve short-term object-recognitionability of spontaneously hypertensive rats: a rodent model of attention-deficithyperactivity disorder. Behav. Pharmacol. 20, 134–145.

Prediger, R.D., Pamplona, F.A., Fernandes, D., Takahashi, R.N., 2005. Caffeine improvesspatial learning deficits in an animal model of attention deficit hyperactivity disorder(ADHD)— the spontaneously hypertensive rat (SHR). Int. J. Neuropsychopharmacol.8, 583–594.

Russell, V.A., Sagvolden, T., Johansen, E.B., 2005. Animal models of attention-deficithyperactivity disorder. Behav. Brain Funct. 15, 1–9.

Sagvolden, T., 2000. Behavioral validation of the spontaneously hyperte`nsive rat (SHR)as an animal model of attention-deficit/hyperactivity disorder (AD/HD). Neurosci.Biobehav. Rev. 24, 31–39.

Segal, D.S., Kuczenski, R., 1987. Individual differences in responsiveness to single andrepeated amphetamine administration: behavioral characteristics and neuro-chemical correlates. J. Pharmacol. Exp. Ther. 242, 917–926.

Sulzer, D., Sonders, M.S., Poulsen, N.W., Galli, A., 2005. Mechanisms of neurotransmitterrelease by amphetamines: a review. Prog. Neurobiol. 75, 406–433.

Tamburella, A., Micale, V., Navarria, A., Drago, F., 2009. Antidepressant properties of the5-HT4 partial agonist SL65.0155: behavioral and neurochemical studies in rats.Progr. Neuropsychopharmacol. Biol. Psych. 33, 1205–1210.

Tsuchida, R., Kubo, M., Kuroda, M., Shibasaki, Y., Shintani, N., Abe, M., Köves, K.,Hashimoto, H., Baba, A., 2009. An antihyperkinetic action by the serotonin 1A-receptor agonist osemozotan co-administered with psychostimulants or the non-stimulant atomoxetine in mice. J. Pharmacol. Sci. 109, 396–402.

Tzavara, E.T., Bymaster, F.P., Overshiner, C.D., Davis, R.J., Perry, K.W., Wolff, M.,McKinzie, D.L., Witkin, J.M., Nomikos, G.G., 2006. Procholinergic and memory en-hancing properties of the selective norepinephrine uptake inhibitor atomoxetine.Mol. Psychiatry 11, 187–195.

Viggiano, D., Ruocco, L.A., Arcieri, S., Sadile, A.G., 2004. Involvement of norepinephrinein the control of activity and attentive processes in animal models of attention deficithyperactivity disorder A.G. Neural Plast. 11, 133–149.

Wilson, W.E., Hudson, P.M., Kanamatsu, T., Walsh, T.J., Tilson, H.A., Hong, J.S., Marenpot,R.R., Thompson, M., 1986. Trimethyltin-induced alterations in brain amino acids,amines and amine metabolites: relationship to hyperammonemia. Neurotoxicol-ogy 7, 63–74.

Woodruff, M.L., Baisden, R.H., Nonneman, A.J., 1991. Anatomical and behavioral sequel-ae of fetal brain transplants performance in rats with trimethyltin-induced neuro-degeneration. Neurotoxicology 12, 427–444.

Yang, P.B., Swann, A.C., Dafny, N., 2006. Chronic methylphenidate modulates locomotoractivity and sensory evoked responses in the VTA and NAc of freely behaving rats.Neuropharmacology 51, 546–556.

Zoli, M., Pich, E.M., Cimino, M., Lombardelli, G., Peruzzi, G., Fuxe, K., Agnati, L.F.,Cattabeni, F., 1990. Morphometrical and microdensitometrical studies onpeptide-and tyrosine hydroxilase-like immunoreactivities in the forebrain of ratsprenatally exposed to methylazoxymethanol acetate. Brain Res. Dev. Brain Res.51, 45–61.

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