Genes, Brain and Behavior (2009) 8: 356–368 # 2009 The AuthorsJournal compilation # 2009 Blackwell Publishing Ltd/International Behavioural and Neural Genetics Society

Methylphenidate to adolescent rats drives enduringchanges of accumbal Htr7 expression: implications forimpulsive behavior and neuronal morphology

D. Leo†,1, W. Adriani*,‡,1, C. Cavaliere§, G. Cirillo§,

E. M. Marco‡, E. Romano‡, U. di Porzio†,

M. Papa§, C. Perrone-Capano†,¶ and G. Laviola‡

†Institute of Genetics and Biophysics ‘A. Buzzati Traverso’, CNR,

Naples, ‡Department of Cell Biology & Neurosciences, Behavioral

Neuroscience Section, Istituto Superiore di Sanita, Rome, and§Faculty of Medicine, Second University of Naples and¶Department of Biological Sciences, ‘Federico II’ University of

Naples, Naples, Italy

*Corresponding author: W. Adriani, Department of Cell Biology &

Neurosciences, Behavioral Neuroscience Section, Istituto Supe-

riore di Sanita’, viale Regina Elena 299 – I-00161 Roma, Italy.

E-mail: walter.adriani@iss.it

Methylphenidate (MPH) administration to adolescent

rodents produces persistent region-specific changes in

brain reward circuits and alterations of reward-based

behavior. We show that these modifications include

a marked increment of serotonin (5-hydroxy-tryptamine)

receptor type 7 (Htr7) expression and synaptic contacts,

mainly in the nucleus accumbens, and a reduction of basal

behavioral impulsivity. We show that neural and behav-

ioral consequences are functionally related: administration

of a selective Htr7 antagonist fully counteracts the MPH-

reduced impulsive behavior and enhances impulsivity

when administered alone in naive rats. Agonist-induced

activation of endogenous Htr7 significantly increases

neurite length in striatal neuron primary cultures, thus

suggesting plastic remodeling of neuronal morphology.

The mixed Htr (1a/7) agonist, 8-OH-DPAT, reduces impul-

sive behavior in adolescent rats and in naive adults, whose

impulsivity is enhanced by the Htr7 antagonist. In sum-

mary, behavioral pharmacology experiments show that

Htr7 mediates self-control behavior, and brain primary

cultures experiments indicate that this receptor may be

involved in the underlying neural plasticity, through

changes in neuronal cytoarchitecture.

Keywords: Animal model, attention deficit/hyperactivitydisorder,delay intolerance, neuralplasticity, nucleus accumbens

Received 3 September 2008, revised 13 January 2009,accepted form publication 19 January 2009

Repeated exposure to psychostimulant drugs causes com-

plex molecular and structural modifications in discrete brain

regions of the mesocorticolimbic dopamine (DA) system, the

main pathway of the brain reward circuitry (Nestler 2005;

Robinson & Kolb 2004; Volkow & Li 2005). This structural

remodeling is similar to other forms of experience-dependent

plasticity, and it is thought to underlie neurobehavioral adap-

tive responses (Hyman et al. 2006; Kelley 2004). The impact

of pharmacological manipulation over DA systems is

enhanced during critical periods of postnatal development,

when this, and other systems, undergo key maturational

changes (Cao et al. 2007, Frantz et al. 2007).

Recently, we have investigated, in rats, the enduringconsequences of adolescent exposure to methylphenidate

(MPH), a psychostimulant drug commonly used for attention

deficit/hyperactivity disorder (ADHD). Such exposure elicited

a long-lasting striatal upregulation (Adriani et al. 2006) of the

kainate 2 subunit of the ionotropic glutamate receptor (Grik2,

also known as GluR6) and of the serotonin (5-hydroxy-

tryptamine, 5-HT) receptor type 7 (Htr7). The striatal complex

is crucial for regulating innate and acquired behavioral habits

and natural reinforcement (Robbins & Everitt 1996) through

its dorsal (i.e. caudate putamen, CPu) and ventral (i.e. nucleus

accumbens, Acb) components, respectively.

The Htr7 is a seven-transmembrane-domain receptor thatis positively coupled to adenylate cyclase (Hedlund & Sutcliffe

2004; Shen et al. 1993) and displays low homology (40%) to

other Gs-coupled 5-HT receptors (Raymond et al. 2001). The

Htr7, localized within the spinal cord (Dogrul & Seyrek 2006),

hypothalamus, hippocampus and (pre)frontal cortex (PFC)

(Neumaier et al. 2001; Ruat et al. 1993; Russo et al. 2005;

Sleight et al. 1995; Venero et al. 1997), has been associated

with several central processes, including learning and mem-

ory (Ballatz et al. 2007; Gasbarri et al. 2008; Hedlund &

Sutcliffe 2004; Meneses 2004; Perez-Garcia & Meneses

2005). Htr7 also modulates DA firing activity in the ventral

tegmental area and plays a prominent role in shaping neuronal

cytoarchitecture (van Kesteren & Spencer 2003; Kvachnina

et al. 2005; Mnie-Filiali et al. 2007). Its central nervous system

distribution together with a high affinity for psychoactive

drugs suggest that Htr7 could arise as a potential therapeutic

target in depression and schizophrenia (Dean et al. 2006; East

et al. 2002; Guscott et al. 2005; Hedlund & Sutcliffe 2007).

The involvement of this receptor in mood regulation and

stress reactivity has been confirmed extensively (Hedlund

et al. 2005; Le Corre et al. 1997; Mullins et al. 1999;

Wesolowska et al. 2006).

We have now explored the neural architecture of the brainreward circuitry in adult rats, following exposure to MPH1Both authors have contributed equally to this work.

356 doi: 10.1111/j.1601-183X.2009.00486.x

during adolescence. We observed a marked increase of Htr7,mainly in the Acb, associated with an increased number of

synaptic contacts. As subchronic MPH exposure in adoles-cent rats also reduces behavioral impulsivity (Adriani & Laviola

2004), an effect that persists into adulthood (Adriani et al.2007; Bolanos et al. 2003), we hypothesized that a functional

link might exist between accumbal Htr7 expression and self-control ability. Therefore, we assessed whether a selective

Htr7 antagonist was able to block the enduring reduction inimpulsivity, resulting from adolescent MPH exposure. We

decided to focus on ‘cognitive’ rather than ‘motor’ impulsivity(Evenden 1999), and therefore we adopted a previously

validated adaptation of delay-discounting task (Adriani &Laviola 2004; Evenden & Ryan 1999) instead of other

protocols, more based on premature and/or non-reinforcedresponding. We hypothesized that the Htr7 receptor could play

its key role in behavioral modulation by means of persistenteffects on neuronal architecture. Thus, we looked for morpho-

logical changes in cultured striatal neurons following adminis-tration of Htr7 agonist and antagonist drugs. Finally, we

investigated the putative role played by Htr1a and Htr7 in basaland pharmacologically modulated impulsive behavior.

Materials and methods

Animal subjects and treatment design

Animal experimental protocols were approved by institutional author-ities, on behalf of Ministry of Health, in close agreement withEuropean Community Directives and Italian Law. All efforts weremade to minimize animal suffering, to reduce the number of animalsused and to use alternatives to in vivo testing. For the first twoexperiments, pregnant Wistar female rats (Harlan, Italy) were housedin an air-conditioned room (temperature 21 � 18C, relative humidity60 � 10%), with a 12-h light/dark cycle (lights on at 0800 h). Waterand food (Enriched Standard Diet; Mucedola, Settimo Milanese, Italy)were available ad libitum. The day of delivery was considered aspostnatal day (PDN) zero, pups being culled to six males and twofemales. Even if only male subjects were used in the presentexperiments, culling included the female gender to avoid carryovereffects of rearing animals in sexual segregation. Pups were thenweaned on PND 21 and housed in groups of two siblings, according tosex. Within litters, each of the siblings was assigned to a different finalgroup. Two separate batches (of eight litters each) were used.

In a first experiment, the consequences of adolescent MPH (Ritalin,CIBA-GEIGY SpA, Milan, Italy) exposure were investigated. Withineach of the eight litters, three pups were assigned to administrationwith MPH (2 mg/kg/day), the other three siblings were assigned toinjection with saline (SAL) during adolescence (PND 30–44). Giventhat learning and memory have significant effects on neuroplasticity,we decided animals for the ex vivo analyses to have no experience atall between last treatment day and the killing. Thus, one SAL and oneMPH sibling per litter (n ¼ 8 rats per group) were killed, at least6 weeks after treatment (PND > 90), for ex vivo analyses. The otherfour animals (two SAL and two MPH sibling per litter, n ¼ 8 rats perfinal group) were tested, at least 3 weeks after treatment(PND > 70), for impulsive behavior.

The latter was measured using the intolerance-to-delay (ID) task. Atthe three highest delays, rats underwent the operant sessionsfollowing challenge with saline vehicle (SAL) or with the Htr7antagonist (R)-3-(2-(2-(4-methyl-piperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)-phenol (SB269970, 3 mg/kg/day for 3 days, 15 min beforethe session; Tocris, Bristol, UK). This was to assess whether long-termMPH consequences could be blocked by a selective Htr7 antagonist.

A second experiment used a second batch of eight litters. Fourmale pups per litter were assigned to another experiment, the othertwo siblings per litter aimed to study the effects of ongoing treatment

witha mixed Htr (1a/7)agonist during theadolescent period (PND 30–44)on impulsivity, as assessed in the intolerance-to-delay task. At thethree highest delays, rats underwent the operant sessions followingchallenge with SAL or with the Htr (1a/7) agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 0.060 mg/kg for 3 days, 30 minbefore the session; Sigma-Aldrich, Milan, Italy). This was to assessif the ongoing effects of MPH, observed in adolescent rats (Adriani &Laviola 2004), were mimicked by a mixed agonist also targeting Htr7.

For the third experiment, Wistar male rats (Harlan) were purchasedwhen adult. At arrival (PND > 60), they were housed in pairs underthe same environmental conditions previously described. After atleast 1 week of acclimation (PND > 70), they underwent the intoler-ance-to-delay task following challenge with either SB269970, the Htr7selective antagonist (or SAL, 45 min before the session), or 8-OH-DPAT, the mixed Htr (1a/7) agonist (or SAL, 30 min before thesession), both at the three highest delays. These naive adult animalswere randomly assigned to one of the four pharmacological treatmentgroups. This experiment aimed to further investigate the role playedby Htr7 in the modulation of baseline impulsive behavior and toinvestigate the participation of Htr7 in the well-known modulatoryeffects of 8-OH-DPAT.

Behavioral impulsivity task

Apparatus and procedureAnimals were tested in an intolerance-to-delay protocol, involvinga choice between a smaller, immediate or a larger, delayed reward(Adriani & Laviola 2004; Evenden & Ryan 1999). Few days before theschedule started, animals were food-restricted to keep them at80–85% of their free-feeding weight, in order to increase theirmotivation to work for food delivery. Each animal was then placeddaily in a computer-controlled operant chamber (Coulbourn Instru-ments, Allentown, PA, USA), provided with two nose-poking holes,a chamber light, a feeder device, a magazine where pellets (45 mg,BioServ, Frenchtown, NJ, USA) were dropped and a magazine light.The nose poking in either hole was detected by a photocell and wasrecorded by a computer, which also controlled food delivery. After the25-min session, animals were returned to their home cage, wherethey were given standard chow (approximately 10 g/each).

During the training phase (1 week), nose poking in one of the twoholes resulted in the delivery of five pellets of food, whereas nosepoking in the other hole resulted in the delivery of one pellet of food.After nose poking and before food delivery, the chamber light wasturned on for 1 second. Following food delivery, the magazine lightwas turned on for 20 seconds, during which nose poking wasrecorded, but was without scheduled consequences (time-out).During the testing phase (1 week), a signaled delay was insertedbetween nose-poking and large-reward delivery. The chamber lightwas kept on during the entire length of this delay. The small rewarddelivery was unchanged. Hence, animals had choice betweena ‘LARGE & LATE’ (LL) or a ‘SMALL & SOON’ (SS) reward. The delaylength was kept fixed for each daily session and was changedprogressively over days. Animals received the corresponding drugchallenge(s) at the three longest delays of this task.

Design and data analysisData were analyzed by split-plot analysis of variance (ANOVA). Thegeneral design of the experiment had two-level drug factors (denotingpretreatment and/or acute challenge with agonist and/or antagonistdrug vs. vehicle) � 4-level delay (set for each session). For Exp. 1 and 2,the litter was always the blocking unit, in that the design comprised allwithin-litter factors. This approach is often used and even recom-mended in studies involving developmental treatments (Zorrilla 1997).In the third experiment, the blocking unit was the subject, and allfactors were between-subject, as adults were purchased as indepen-dent individuals. Multiple comparisons were then performed withTukey HSD when allowed.

The test phase consisted indeed of two halves: (1) the first foursessions, when delays were introduced but animals were allowed toface them without acute drug challenge and (2) the last four sessions,at the highest delays, including a baseline reference point plus the lastthree sessions, when the acute challenge was indeed administered.

Genes, Brain and Behavior (2009) 8: 356–368 357

Long-term consequences of methylphenidate during adolescence

ANOVAs to evaluate drugs’ effects were conducted on the second half.Separate analyses were performed within levels of a given factorwhen allowed by a main effect of that factor.

The first dependent variable considered was the choice (%) for thelarge reinforcer, namely percentage of LL over total LL þ SS choices,during the test phase. The second variable considered was the slopeof the preference–delay curve, calculated using MICROSOFT EXCEL

‘slope’ function, with the large-reward preference as y-axis data andlog (delay þ 1) as x-axis data (Evenden & Ryan 1999). Criteria forexclusion were: large-reward preference lower than 50% at the endof the training phase, or large-reward preference at highest delayhigher than that shown at delay ¼ 0. Accordingly, in Exp. 3, a total ofthree rats were excluded from data analyses.

Brain markers and morphology

The animals assigned to ex vivo analyses were killed at a point whenthe corresponding siblings had completed the behavioral testing.Three rats per group were randomly assigned to the brain dissectionand five rats per group were assigned to the brain perfusion.

Western blot analysisAfter killing, the brain was quickly dissected on dry þ wet ice. Thewhole-striatal complexes were collected bilaterally and used forWestern blot analysis. Brain striatal hemispheres were weighed,homogenized, split in two halves and pooled. To the purpose of usingsix final samples per group, we prepared six independent poolscoming from two contra-lateral hemispheres taken from two differentanimals out of the three. Striatal proteins were obtained using theTri-Reagent isolation system, following manufacturer’s instructions.Total protein concentration was determined by Bradford method(Bradford 1976). To generate Western blots, 25 mg of protein/lanewas loaded onto 10% SDS-polyacrylamide gels and resolved usinga mini-Protean II (Bio-Rad Laboratories) gel apparatus for 1 h at 100 V.A molecular weight standard was also included on the gel (Rainbowstandards; Bio-Rad Laboratories). Proteins were electrophoreticallytransferred onto polyvinylidene difluoride membranes (Immobilon-P,Millipore, Watford, UK) with a semi-dry transblotting apparatus. Themembranes were blocked in 5% (w/v) non-fat milk in Tris-bufferedsaline Tween-20 (TBST) (0.1% Tween, 150 mM NaCl, 10mM Tris-HCI,pH 7.5) and incubated with primary antibodies for 1 h at roomtemperature. After washing in TBST, membranes were incubatedfor 1 h at room temperature with HRP-conjugated secondary anti-bodies (dilution 1:1000; Bio-Rad) in TBST buffer containing 5% non-fatmilk. The blots were then washed three times for 15 min in Tris-buffered saline, and exposed to Kodak film using ECL chemilumines-cence (GE Health Care, Milan, Italy). Finally, the blots were strippedand probed with a b-actin antibody. The bands from the Western blotswere densitometrically quantified on X-ray films using a software(IMAGEQUANT) to determine the levels of immunoreactivity. Monoclonalmouse antibodies to tyrosine hydroxylase (TH) (#MAB318, 1:500;Chemicon, Milan, Italy), polyclonal rabbit antibodies to Grik2 (#06-315;1:200; Upstate Biotechnology Inc., Lake Placid, NY, USA), polyclonalrabbit antibodies to Htr7 (#PC249L, 1:500; Calbiochem, Milan, Italy),and monoclonal mouse b-actin antibodies (#A2668, 1:5000; Sigma-Aldrich, Milan, Italy) were used. Data from bands corresponding to themolecular weight of the detected protein were normalized to b-actin. Thedensity in the MPH group was then expressed as fold change over theSAL group, set as reference level at 100%.

Tissue preparationAfter killing, the brains of perfused animals were removed (fiveanimals per group). Rats were deeply anesthetized with a chloralhydrate (300 mg/kg i.p.) and perfused transcardially with a solution(Tris–HCl 0.1 M, ethylenediaminetetraacetic acid (EDTA) 10 mM) fol-lowed by 4% paraformaldehyde plus 0.1% glutaraldehyde in 0.01 M

phosphate-buffered saline (PBS), pH 7.4 at 48C. For light microscopy,the brain was removed and post-fixed 2 h in the same fixative, thensoaked in 30% sucrose PBS and frozen in chilled isopentane on dryice. Serial sections were cut on a cooled sliding microtome ata thickness of 25 mm and collected in cold PBS for immunocytochem-

istry. For electron microscopy, the brain was removed and placedovernight in 4% paraformaldehyde added to 1% glutaraldehyde in0.01 M PBS, pH 7.4 at 48C. Then, the tissue was rinsed in phosphatebuffer and post-fixed in 2% osmium tetroxide in phosphate buffer for20 min, dehydrated in ethanol and embedded in araldite resinembedding kit (TAAB Laboratories Equipment Ltd, AldermastonBerkshire, UK). Ultra-thin sections, then, were stained with uranylacetate and lead citrate and examined with an EFTEM Lybra 120electron microscope (Zeiss, Oberchoken, Germany).

ImmunocytochemistryBrain sections were blocked in 10% normal serum in 0.01 M PBS,0.25% Triton for 1 h at room temperature. Each primary antibody wasdiluted in 0.01 M PBS containing 10% normal serum 0.25% Triton. Free-floating slices were incubated for 48 h at 48C on an oscillating plate.Sections were washed several times in PBS and incubated with theappropriate biotinylated secondary antibody (Vector Labs Inc., Burlin-game, CA, USA; 1:200) for 90 min at room temperature, washed in PBSand processed using the Vectastain avidin–biotin peroxidase kit (VectorLabs Inc.) for 90 min, also at room temperature. The sections werewashed in 0.05 M Tris–HCl and reacted with 3,3-diaminobenzidinetetrahydrochloride (DAB; Sigma, Milan, Italy) in 0.5 mg/ml Tris–HCland 0.01% hydrogen peroxide. Sections were mounted on chrome-alume–gelatine-coated slides, dehydrated and coverslipped. Adjacentsections were Nissl-stained. Slides were imaged with a Zeiss Axio-skope 2 light microscope (Zeiss) equipped with high-resolution digitalcamera (C4742-95, Hamamatsu Photonics, Milan, Italy).

A densitometry of the several markers and morphometric featuresof cells in CPu, Acb and PFC was accomplished using computer-assisted image analysis system (MCID 7.0; Imaging Res. Inc.,Ontario, Canada). Sections of the CPu were analyzed within theinterval þ2.20 to �1.30 from bregma (Paxinos & Watson 2001). TheCPu was divided in two halves a dorsolateral (Dl-) and ventro-medial(Vm-) one. The Acb was outlined for each section and analyzedaccording to Nissl-stained adjacent sections and the calbindin-treatedsections of Zahm and Brog (1992). The core was divided from theshell throughout the posterior horn of the anterior commissure.Sections of the PFC were analyzed within the interval þ5.20 toþ3.20 from bregma (Paxinos & Watson 2001).

For Htr7 and TH expression, where labeling gave a diffuse appear-ance, we measured the mean density (density � area) within theselected regions. For Substantia P SubP and Grik2 expression, wherelabeling allowed discrete imaging of structures, we counted thenumber of positive cells elements, and therefore these two markerswere expressed as proportion of the total target area relative to thescanned area. Data were obtained from 10 randomly selectedsections for each region and then averaged for treated (MPH) andfor saline control (SAL) group.

Electron microscopyFor electron microscopy analysis, to estimate the synaptic density andactive zone length in the nucleus accumbens (Schikorski & Stevens1997), we choose a previously reported systematic sampling methodto reduce sampling bias (French & Trotterdel 2004). This wasachieved by collecting five sections per grid on three grids and then4 mm of the block was removed and the collection repeated; thisprocedure was repeated eight times. This technique allows to samplethroughout the depth of the sample. The middle section of the middlegrid in each collection of three grids was photographed at 42 000�magnification. In an alternative pattern, on one section we acquiredfour pictures proceeding from the top left corner to the bottom rightcorner; on the following section, we inverted direction of acquisitionfrom the top right corner to the bottom left one. Then synapticcomplexes were quantified. Synaptic complexes were defined asstructures that met two criteria: (1) restricted zone of two membranesin apposition, one or both exhibiting increased thickness or electroncontrast and (2) presence of vesicles, 30–50 nm in size, in one or bothof the two cytoplasmic domains separated by the opposing mem-branes. Digital images were analyzed using computer-assisted imageanalysis system (MCID 7.0; Imaging Res. Inc.).

All data collection was performed blind, with the observer being notaware of the treatment groups. These data were exported and

358 Genes, Brain and Behavior (2009) 8: 356–368

Leo et al.

converted to a frequency distribution histogram using the SIGMA-PLOT

v8.0 program (SPSS, Erkrath, Germany). Individual images of controland treated rats were assembled, and then the same adjustmentswere made for brightness, contrast and sharpness using ADOBE

PHOTOSHOP (Adobe Systems, San Jose, CA, USA).

Neuronal primary cultures

PreparationTimed pregnant Sprague–Dawley rats (Charles River, Italy) were killedin accordance with Society for Neuroscience guidelines. The embry-onic age (E) was determined by day of insemination (i.e. the ap-pearance of vaginal plug was considered as day E0). About 25–30embryos from different dams were pooled at E16 and were used forpreparation of four culture dishes.

The striatum (STR) was quickly dissected from embryos, undera stereoscope in sterile conditions, and placed in PBS withoutcalcium and magnesium and supplemented with 33 mM glucose.Cells were dissociated from embryonic STR and cultured as pre-viously described (di Porzio et al. 1980). Briefly, the dissectedE16 striata were enzymatically dissociated by incubation for30 min at 378C in a papain solution (Warthington, 20 U/mL) in Earle’sbalanced salts containing 1 mM EDTA, 1 mM cysteine and 0.01%pancreatic DNAse. After addition of 1 mg/mL of bovine serumalbumin (fraction V, Sigma-Aldrich) and 1 mg/mL ovomucoid(Sigma-Aldrich) the cell suspension was centrifuged 10 min at800 g, suspended in plating medium with a fire-polished Pasteurpipette and counted (Fiszman 1991).

Cells were plated at a density of 300 000/cm2 (Lab-Tek� ChamberSlides�, Nunc, Milan, Italy), in four dishes coated with 15 mg/mL ofpoly-D-lysine (Sigma). Cells were grown in neurobasal medium(Invitrogen, Milan, Italy), supplemented with B27 (Invitrogen). Cul-tures were maintained at 378C in a humidified incubator for 3 days.The cell cultures were treated with 100 nM of the Htr (1A/7) receptoragonist (8-OH-DPAT, Sigma-Aldrich, Milan, Italy), or with 100 nM ofthe Htr7 antagonist (SB269970, Tocris, Bristol, UK), or with a combi-nation of these two drugs. Agonists of D1 or D2 DA receptors (i.e.either deoxyepinephrine HCl or Quinpirole, respectively; Sigma) werealso administered. Drugs were added to cultures 48 h after cell platingand the drugs were left in culture medium for 24 h.

Measurement of neurite outgrowth and numberCell cultures were fixed for 30 min at room temperature, in 4%paraformaldehyde in PBS, followed by three washes in PBS, per-meabilized for 15 min in PBS containing 0.1% Triton-X-100 and 10%normal goat serum (NGS) and incubated for 2 h at room temperatureor overnight at 48C in the primary antibodies diluted in PBS containinga 10% of NGS. We used the monoclonal antibody (mAb) againstneuronal class III-tubulin (anti-Tuj1; 1:750, Chemicon). After incuba-tion with the primary antibody, cell cultures were rinsed in PBS andwere either stained according to standard avidin–biotin immunocyto-chemistry procedures (Vectastain Elite, Vector Labs Inc.) usingperoxidase substrate kit (dia-aminobenzidine) or incubated withfluorescent-labeled secondary antibodies (Texas-red goat anti-rabbit,Molecular Probes Inc., Eugene, OR, USA; goat anti-mouse fluores-cein-conjugated, Chemicon) in PBS containing a 5% solution of NGS.Control cells were incubated in the same solutions without primaryantibodies and subsequently processed as above.

After Tuj1 staining, cell-culture slides were analyzed by a ZeissAxioskop 20 XYZ motorized stage (Prior Proscan II) microscope usingthe software MCID Elite High Definition analog Camera HamamatsuC5405. The MCID Elite imaging system (Imaging Research Inc.,Linton, UK) was used for imagine acquisition and operator-controlledtracing of neuritis. A total of 12 fields for each cell-culture conditionwere selected from four independently treated culture wells, eachcontaining from 4–10 cells. The number, total and mean lengths ofbranches per neuron were measured.

Statistical analysesData were analyzed using the Student’s t-test for both immunohisto-chemical (Fig. 1a–d) and electron microscopy (Fig. 2) analyses, for

quantification of proteins in Western blot analyses (Fig. 1e) and formeasurement of neurite outgrowth (Fig. 6b,c). A difference wasaccepted as significant if the probability level was less than 5%(P < 0.05).

Results

Neuronal markers and cytoarchitecture in brain

reward pathways are altered by MPH

Rats, exposed to MPH (2 mg/kg/day) or saline (SAL) duringadolescence, at postnatal days (PND) 30–44, were used at

adulthood either for the behavioral studies (PND 70) or for theneurochemical and morphological analyses (PND 90). We first

examined, in samples coming from the whole-striatal com-plexes, protein levels of two neurotransmitter receptors,

Grik2 and Htr7, whose mRNA transcripts were found to bepersistently upregulated following adolescent MPH exposure

(Adriani et al. 2006). Western blot analyses showed that the

levels of both proteins were significantly higher in the whole-striatal complex of adult rats as a consequence of MPH

exposure during adolescence, when compared with SALcontrols (P < 0.05; Fig. 1a,b).

We also analyzed the protein levels of the glutamic aciddecarboxylase (GAD65) and the tyrosine hydoxylase (TH),

two key neuronal enzymes. GAD65 was investigated asa marker for g-aminobutyric acid (GABA)ergic neurons that

constitute the majority of striatal neurons. TH was studied asit labels catecholaminergic fibers, most of which are dopami-

nergic terminals of the mesocorticolimbic pathway (Graybiel1990). The protein levels of both enzymes were significantly

higher in the adult striatal complex as a consequence of MPHexposure during adolescence, when compared with SAL

controls (P < 0.05; Fig. 1a,b).To map the anatomical localization of the Grik2 and Htr7

increase in target forebrain areas of the mesocorticolimbicpathway, we performed immunohistochemical analyses.

Grik2 showed more intense staining in the dorsolateral (Dl-)and ventromedial (Vm-) Cpu and in the PFC of the MPH group,

compared with the SAL group. In both core and shell ofthe Acb (AcbC and AcbSh), the densitometric analysis for

Grik2 expression did not show differences between the twogroups (Fig. 1d). As for the Htr7 immunoblotting data, the

densitometric value in the Acb and in the PFC was signifi-cantly higher for MPH-exposed animals, compared with the

SAL group. In the CPu, however, the Htr7 staining wascomparable in both groups (Fig. 1c). We also analyzed by

immunohistochemistry the expression of Substantia P (SubP)and TH. SubP was investigated as it is expressed by a subset

of GABAergic neurons that constitute the direct striatal out-put pathway (Gerfen 1988). The SubP and the TH staining

were both significantly more intense in the Dl-CPu and in the

PFC of MPH-exposed rats than in the SAL group. In theVm-CPu and in both core and shell of Acb, SubP and TH

staining did not show any difference between the two groups(Fig. 1e,f).

In summary, we observed region-specific long-term con-sequences of an adolescent MPH exposure in selected

forebrain areas. All of the markers were increased in thePFC. Among these markers SubP, TH and Grik2 were altered

Genes, Brain and Behavior (2009) 8: 356–368 359

Long-term consequences of methylphenidate during adolescence

in the CPu but not in the Acb. Conversely, Htr7 was indeedthe only marker showing a persistent MPH-induced modifi-

cation in the Acb but not in the CPu. Such striking modifica-tion in accumbal Htr7 levels, produced by adolescent MPH,

could be implicated in plastic rearrangement within thisregion, which was hence chosen for deeper analyses of

synaptic plasticity. Indeed, we evaluated possible enduringchanges in active zone length and in synaptic number and

shape, as detectable measures of neurotransmitter releaseand therefore as indexes of synaptic plasticity.

We thus counted asymmetric synapses in random fields ofAcb (Fig. 2a,b). MPH-exposed animals had a higher synaptic

density compared with SAL controls (CTR) (3.85 � 0.24synapses/9 mm2 of neuropil vs. 2.96 � 0.16 synapses/

9 mm2; P < 0.01; Fig. 2c). We then measured the active zonelength, defined as a specialization of the presynaptic mem-

brane thought to be the site of synaptic vesicle exocytosis.The length of the active zone measured in asymmetric spine

synapses found in a random sample of neuropil fields withinthe Acb was slightly, although significantly, shorter in MPH-

exposed rats, compared with SAL controls (CTR)(192.96 � 4.28 vs. 212.52 � 5.1 mm; P < 0.01; Fig. 2d).

Neurite outgrowth is induced by Htr7 stimulation in

cultured striatal neurons

Htr7 has been recently involved in behavioral impairment

(Guscott et al. 2005; Hedlund et al. 2005; Roberts et al. 2004),in mood regulation (Bonaventure et al. 2002; Wesolowska

et al. 2006), in mnemonic processes and cognition (Ballatzet al. 2007; Cifariello et al. 2008; Gasbarri et al. 2008) and in

regulating cell cytoarchitecture (Kvachnina et al. 2005). Wethus hypothesized that Htr7 may modulate behavioral impul-

sivity via plastic changes at the neuronal level. To evaluate theeffects of Htr7 activation on neuronal morphology and neurite

outgrowth, we used primary cultures obtained from thestriatal complex of rats at the embryonic day (E) 16. Virtually

all cells in our cultures expressed the neuronal Tuj1 marker, asshown by coexpression with the nuclear marker DAPI, thus

indicating that the majority of cultured cells were neurons. Inaddition, more than 90% of striatal neurons were GABAergic

(as shown by immunoreactivity to the enzyme glutamatedecarboxylase) and were immunoreactive for the Grik2 sub-

unit and for Htr7 (Fig. 3a). Striatal cultures were treated for24 h with the appropriate agonist and/or antagonist, stained

with the anti-Tuj1 antibody (Fig. 3b) and analyzed

Figure 1: Quantitative analysis of

selected neuronal proteins in the

brain of MPH-exposed rats. (Panels

a,b) Western blot analyses performed

on the striatal complex of adult male rats

(PND 90) exposed to MPH (2 mg/kg/day)

or SAL during adolescence (PND 30–44).

A representative example of blots stained

with antibodies against GAD65, TH, Grik2,

Htr7 and b-actin (a) is shown. The density

of each protein, normalized to b-actin

staining, was expressed for the MPH

group (b) as mean � SEM fold change

over the respective SAL group, set as

reference level at 100% (Student’s t-test,

n ¼ 6; *P < 0.05). (Panels c–f) Quantifica-

tion of immunocytochemical staining with

antibodies against Htr7 (c), Grik2 (d), Sub

P (e) or TH (f) in different brain regions (i.e.

PFC, Dl-CPu, Vm-Cpu, both AcbC and

AcbSh) of adult male rats (PND 90)

exposed to MPH (2 mg/kg/day) or SAL

during adolescence (PND 30–44). The

expression of Htr7 (c) or TH (f) was mea-

sured by the mean density (density �area). In the same regions we counted

the number of cells positive for Grik2 (d) or

SubP (e), therefore these markers are ex-

pressed as proportion of the total target

area relative to the scanned area (propor-

tional area). The data are obtained from ten

randomly selected sections for each

region (Student’s t-test, n ¼ 5; *P <

0.05, **P < 0.01).

360 Genes, Brain and Behavior (2009) 8: 356–368

Leo et al.

morphometrically. Exposure of cultures to 8-OH-DPAT, the

mixed Htr (1a/7) agonist, resulted in a significant increase inthe total length (Fig. 3c) and mean length (data not shown) of

neurites, when compared with untreated control cultures(CTR, set to 100%). These effects were at least partially

induced by selective stimulation of the Htr7 because theywere completely blocked by the simultaneous treatment with

the highly selective Htr7 receptor antagonist SB269970.Treatment with the Htr7 receptor antagonist alone had no

effect on the total length of neurites. Other morphological

parameters, such as the number of neurites, the end nodes,the branch nodes, the soma perimeter and the total area of

each neuron were not affected by stimulation of the cultureswith 8-OH-DPAT (data not shown).

Treatment with agonists of D1 or D2 DA receptors did notaffect any parameter in the morphology of striatal cultures

(data not shown). These results further emphasize theselective role of Htr7 activation in the outgrowth of striatal

neurites, as stimulation of other abundantly expressedG-protein-coupled receptors was ineffective.

Modulation of behavioral impulsivity by

Htr7-targeting drugs

Behavioral impulsivity can be studied in laboratory settings,

based on the assumption that impulsive subjects are lesstolerant to (and feel aversion for) situations of delayed reward

(Adriani & Laviola 2004; Evenden 1999; Evenden & Ryan1999). Adult animals, exposed to either MPH or SAL during

adolescence, were given choice between the immediatedelivery of one pellet of food (small and soon, SS), or the

delivery of five pellets of food after a delay (large and late, LL)

that was increased progressively over subsequent dailysessions (see Materials and methods). As expected, all rats

expressed significant preference for LL at the end of thetraining phase, when LL had no delay, then started to shift

toward SS choices (delay F3,33 ¼ 5.57, P < 0.01), as delayprogressively increased during the first half of the test phase.

To evaluate if a selective Htr7 blockade could counteract theMPH-reduced impulsivity, animals were assessed with the

selective Htr7 antagonist, SB269970, or its saline vehicle (SAL)

on the second half of the test phase, when LL choices were stillprogressively decreasing (delay F3,33 ¼ 9.02, P < 0.001). Con-

sistent with previous work (Adriani et al. 2007), a reduced levelof impulsivity appeared as a long-term consequence in MPH-

exposed subjects, compared with SAL-exposed controls (pre-treatment, F1,11 ¼ 8.56, P < 0.05), especially at the 45- and

60-second delays (Fig. 4). As the SAL and MPH pretreated ratsdo differ considerably, as we expected based on our previous

work (Adriani et al. 2007), we had no possibility of formulatinga null hypothesis for a general ANOVA, i.e. to assume that these

two pretreatment groups do not differ. The two pretreatmentgroups were hence analyzed separately.

In response to the Htr7 antagonist SB269970, no significanteffects were evident within the SAL-exposed group (challenge,

F1,6 ¼ 0.05, not significant). Conversely, within the MPH-exposed group, rats challenged with the Htr7 antagonist

exhibited significantly lower LL choices, compared with theircorresponding SAL controls (challenge, F1,5 ¼ 7.79, P < 0.05),

at the 45- and 60-second delays (Fig. 4b). In summary, Htr7antagonist administration per se failed to exert consistent

effects, at least in rats that were born in our facility, were

Figure 2: Synaptic organization in the

Acb of MPH-exposed rats. Changes in

the number and structure of synaptic

contacts in adult male rats (PND 90)

exposed to MPH (0 or 2 mg/kg/day) during

adolescence (PND 30–44). The synaptic

complexes are shown (arrow) for SAL (a)

and MPH (b) exposed groups (individual

sampling area, 9 mm2; total sampling area,

270 mm2). (c) The number of Acb synapses

shows a significant increase (Student‘s

t-test, n ¼ 5; **P < 0.01) in MPH-exposed

rats (filled column) (3.85 � 0.24 synapses/

9 mm2) compared with controls (CTR, open

column) (2.96 � 0.16 synapses/9 mm2). (d)

The active zone (AZ) length (mm) of the

accumbal synapses (post-synaptic density)

shows a significant decrease (Student’s

t-test, n ¼ 5; **P < 0.01) in MPH-exposed

rats (filled column) (192.96 � 4.28 mm)

compared with controls (CTR, open col-

umn) (212.52 � 5.1 mm). Scale bar:

0.5 mm.

Genes, Brain and Behavior (2009) 8: 356–368 361

Long-term consequences of methylphenidate during adolescence

handled during adolescence and were then assessed weekslater. Interestingly, the Htr7 antagonist was fully able to

counteract the persistent profile of reduced impulsivity, gener-ated asa long-term consequence byadolescent MPH exposure.

A separate group of adolescent rats without any priorpretreatment were tested for modulation of impulsive behav-

ior by Htr7 stimulation. Rats were challenged with the mixedHtr (1a/7) agonist 8-OH-DPAT or its vehicle on the second half

of test phase, when LL choices were still progressivelydecreasing across sessions (delay F3,27 ¼ 6.01, P < 0.01).

Interestingly, LL choices were significantly higher in drug-injected rats than in corresponding controls (challenge �delay interaction, F3,27 ¼ 2.94, P < 0.05 ; Fig. 5), specificallyat the 75-second delay. These findings show the capacity of

this mixed Htr (1a/7) agonist to reduce impulsivity, i.e. topositively modulate behavior at least when basal levels of

impulsivity are enhanced, as is typical of the adolescent phase(Adriani & Laviola 2003; Laviola et al. 2003). In this, 8-OH-

DPAT seemed to mimic the therapeutic effects onimpulsivity, produced with an ongoing MPH treatment during

adolescence (Adriani & Laviola 2004).As 8-OH-DPAT is known to act as both Htr (1a) and Htr (7)

agonist, we aimed to investigate the specific role of Htr7 inthe anti-impulsive effects of 8-OH-DPAT. Naive adult animals

were challenged on the last three test sessions, when LLchoices were progressively decreasing (delay F3,84 ¼ 17.68,

P < 0.0001), with the selective Htr7 antagonist SB269970 incombination with the mixed Htr (1a/7) agonist 8-OH-DPAT.

Data from slopes of the preference curves (Table 1) yieldedan interaction between SB269970 and 8-OH-DPAT (i.e.

antagonist � agonist, F1,28 ¼ 4.22, P < 0.05; Fig. 6 andTable 1). Multiple comparisons showed no significant effects

of 8-OH-DPAT alone among adult animals. Conversely, theHtr7 antagonist per se increased impulsivity levels in naive

rats, thus suggesting a role for this receptor in modulatingbasal impulsivity. The slight discrepancy on the effects of the

Figure 3: Neurite outgrowth induced by Htr7 stimulation in cultured striatal neurons. (a) Photomicrographs of striatal (STR)

cultures. Most of the cells in primary striatal cultures coexpress the nuclear marker DAPI (blue) and the neuronal marker Tuj1 (red), the

GABAergic marker GAD6 (green), the glutamate receptor subunit Grik2 (red), or the Htr7 (green). Scale bar, 100 mm. (b) Effects of Htr7

signaling on neuronal morphology in vitro. Immunostaining for Tuj1 antibody on STR cultures, treated for 24 h with agonist/antagonist of

Htr7 and/or Htr1a. CTR, control cultures; ANT, STR cells treated with 100 nM SB269970, a selective Htr7 antagonist; AGO, STR cells

treated with 100 nM 8-OH-DPAT, a Htr (1a/7) agonist; AGOþANT, STR cells treated with 100 nM SB269970 and 100 nM 8-OH-DPAT

(Student t-test; n ¼ 4; *P < 0.05). (c) Regulation of neurite length by Htr7 in STR cultures. Total neurite length was measured on STR

cultures stained with anti-Tuj1 antibody by MCID Elite imaging system (see Methods). Cells were treated for 24 h with: 100 nM

SB269970, the selective Htr7 antagonist; 100 nM 8-OH-DPAT, the Htr (1a/7) agonist; 100 nM SB269970 and 100 nM 8-OH-DPAT. Data

were expressed as a percentage of untreated control cultures, set to 100%. The bars represent means � SEM from randomly selected

fields for each cell-culture condition (Student t-test; n ¼ 4; *P < 0.05).

362 Genes, Brain and Behavior (2009) 8: 356–368

Leo et al.

antagonist per se may be ascribed to uncontrolled effects ofprevious handling for drug exposure during adolescence.

Our findings clearly show the capacity of a mixed Htr (1a/7)agonist to positively modulate behavior in the presence of

enhanced basal levels of impulsivity, which were found innaive adult animals treated with the Htr7 antagonist. In this

group, indeed, the coadministration of 8-OH-DPAT reducedimpulsivity levels, by counteracting the pro-impulsive effects

of the Htr7 antagonist alone. Present results confirm the Htr7as an important player in the modulation of basal levels of

impulsive behavior, suggesting a potential benefit from this

receptor as a target for novel therapy in self-control disorders.

Discussion

We have recently reported that MPH produces differential

brain effects during adolescence, compared with adult rats(Canese et al. 2009). Adolescent MPH exposure modulates

gene expression in the striatal complex, suggesting itslong-lasting impact on many neuronal functions: Notably, a

persistent upregulation of striatal Htr7 mRNA was associatedwith a low basal level of behavioral impulsivity (Adriani et al.

2006 2007). We are now reporting that increased expression

Figure 4: Blockade of adolescentMPH consequences on impulsivity by the Htr7 antagonist, SB269970. Mean (�SEM) choice (%)

of the large but delayed reinforcer, by nose poking at the large/late hole, shown in drug-free state by adult male rats (PND 70) exposed to

MPH (0 or 2 mg/kg/day) during adolescence (PND 30–44). First five sessions were pooled, as the challenge (SB269970 or SAL) was not

yet administered to rats. Impulsivity was reduced as a long-term consequence of adolescent MPH (Tukey post hoc tests between

adolescent exposure groups, n ¼ 8; *P < 0.05), as shown on saline vehicle (SAL) challenge (white circles). Impulsivity levels, shown by

MPH-exposed rats (b) on SB269970 (3 mg/kg once/day) challenge (black circles) were drawn to those expressed by the SAL-exposed (a)

control groups (Tukey post hocs between challenge groups, n ¼ 8; *P < 0.05).

Figure 5: Acute effects of the Htr (1a/7) agonist, 8-OH-DPAT,

on impulsivity in adolescent rats. Mean (�SEM) choice (%) of

the large but delayed reinforcer, by nose poking at the large/late

hole, shown by adolescent (PND 30–44) male rats born in our

colony. The first five sessions were pooled, as no challenge was

yet administered. Rats were challenged with 8-OH-DPAT (0 or

0.06 mg/kg once/day) during the last three sessions at the

longest delay (Tukey post hoc tests; n ¼ 8; *P < 0.05).

Table 1: Effects of 8-OH-DPAT and SB269970 combination on

impulsive behavior in naive adult rats

Group Slope value

Vehicle Vehicle �23.97 � 2.48

Vehicle 8-OH-DPAT �25.22 � 2.08

SB269970 Vehicle �32.36 � 2.31*

SB269970 8-OH-DPAT �22.97 � 3.33†

Slope (mean � SEM) of the preference–delay curve from naive adult

(PND 70) male rats receiving two drugs (first injection: SB269970 or

SAL; drug challenge: 8-OH-DPAT or SAL) on the last 3 days of the

intolerance-to-delay task (Tukey post hoc tests, n ¼ 8). *P < 0.05 vs.

vehicle-vehicle animals; †P < 0.05 vs. SB269970-vehicle animals.

Genes, Brain and Behavior (2009) 8: 356–368 363

Long-term consequences of methylphenidate during adolescence

of Htr7 occurs mainly in the Acb and is accompanied by long-lasting changes in synaptic number and shape, detected up to

3 months after the end of the MPH treatment. In addition, wereport a key role for Htr7 in the basal modulation of impulsive

behavior, possibly via remodeling of neuronal morphology, aswe show in vitro. Among the persistent consequences of

adolescent MPH exposure, we also observed region-specificchanges of selected neuronal markers within key areas of the

brain reward circuits. In particular, an increase in the immu-noreactivity for SubP, TH and Grik2 was specifically localized

within the CPu. These results indicate that adolescent MPH

treatment causes adaptations in neurons of the direct striataloutput pathway as shown by the increased expression of the

neuropeptide SubP (Gerfen 1988).There is now evidence that, following administration of

psychostimulant drugs, firing of dopaminergic afferents fromthe midbrain and glutamatergic afferents from the cortex

converge on the same neurons (e.g. GABAergic medium-sized spiny neurons within the striatum or pyramidal cells

within the cortex), which act as coincidence detectors andplay a key integrative role in modulating motivation and

adaptive behavior (Kelley 2004; Mohn et al. 2004; Zhanget al. 2006). Our results, showing increased TH and Grik2

immunoreactivity not only in the CPu, but also in the PFC,suggest that adolescent MPH treatment can alter the sub-

strate circuitry for integration of dopaminergic and glutama-tergic neurotransmission both in the striatum and in the PFC,

two crucial components of the reward circuitry network. Ourresults are in agreement with reports showing MPH-induced

alterations in several molecular markers, preferentially in thesensorimotor striatum (Yano & Steiner 2005).

The Htr7 was the only marker to be selectively and stronglyupregulated in the Acb, a key area of the brain reward circuit,

involved in the processing of primary natural reinforcement(Robbins & Everitt 1996) and in the rewarding effects of drugs

(Hernandez & Kelley 2005; Ikemoto et al. 2005). The adminis-tration of MPH during adolescence determined an increased

number of synaptic contacts and a reduction of the active zone,which were detected in the Acb 3 months later. These long-

lasting effects indicate an enduring remodeling of synapticmorphology in this brain area. While it is known that morpho-

logical remodeling of dendritic spines follows the administra-tion of cocaine, amphetamine and nicotine (Hernandez & Kelley

2005; Robinson & Kolb 2004), to our knowledge this is the firstdemonstration that MPH can induce persistent changes in the

structure of striatal synapses, similarly to other psychostimu-

lants. The dorsal and ventral striata (CPu, Acb) play importantroles for mammalians: Acb DA enables salient stimuli repre-

sentations to acquire incentive properties, and facilitatesa flexible approach response (Berridge 2007; Berridge &

Robinson 1998; Salamone et al. 2003), in contrast to the fixedinstrumental approaches (i.e. habits) which rather involve the

nigro-striatal DA system (Ikemoto & Panksepp 1999). More ingeneral, forebrain DA and 5-HT play a central role in psycho-

motor control, including subcortical processing of behavioralpatterns and cortical processing of inhibitory control (Ragozzino

2003; Ridderinkhof et al. 2004).On the other hand, the persistent increase of Htr7 expres-

sion in response to chronic MPH is noteworthy. MPH is ableto increase extracellular concentrations of DA and noradren-

aline, but not 5-HT, in the brain (Kuczenski & Segal 1997).However, recent studies have extended to the 5-HTergic

system a role in ADHD pathogenesis as well as a contributionto the putative mechanisms of MPH therapeutic effects.

Indeed, striking 5-HT-system changes, like striatalhyper-innveration as well as increased expression of 5-HT

transporter and receptor subtypes, are reported in ADHDanimal models (Descarries et al. 1992), also supporting the

anti-hyperactivity effects of selective 5-HT reuptake inhibitors(SSRIs) (Davids et al. 2002). Moreover, strong synergistic

Figure 6: Effects of 8-OH-DPAT and SB269970 combination on impulsive behavior in naive adult rats. Mean (�SEM) choice (%)

of the large but delayed reinforcer, by nose poking at the large/late hole, shown by adult (PND 70) naive male rats. First five sessions were

pooled, as drug combinations were not yet administered. Rats then received two drugs (first injection: SB269970 or SAL; second

injection: 8-OH-DPAT or SAL) before the last three sessions. (a) Among SAL-injected rats, no significant effect of 8-OH-DPAT was

evident. (b) SB269970 significantly enhanced the impulsive behavior of rats (black circles), whereas 8-OH-DPAT reversed the impulsive

profile exhibited by Htr7 blockade in SB269970-injected animals (black triangles), possibly acting as a Htr1a agonist (n ¼ 8; see also slope

values in Table 1).

364 Genes, Brain and Behavior (2009) 8: 356–368

Leo et al.

interactions exist between MPH and 5-HT (1B) receptoragonists (Borycz et al. 2008), and treatment with fluoxetine,

a classical SSRI, can normalize adult behavioral deficitsinduced by early-life exposure of rats to MPH (Bolanos et al.

2008). These findings suggest a functional link betweenADHD, the effects of MPH, and the 5-HTergic system. Here,

we extend these putative interactions, highlighting thata strong and enduring Htr7 upregulation (following adolescent

MPH administration) has functional implications for impulsivebehavior, and is consistent with the known interactions

between Htr7 and neural effects induced by psychostimulantdrugs of abuse (Mnie-Filali et al. 2007).

The neurophysiological mechanism responsible for theseeffects is still unknown. As for MPH-induced Htr7 upregulation,

it can be speculated that MPH-increased catecholamine levelscould trigger a cascade of adaptive events, leading to a long-term

Htr7 increase. As for the role of Htr7 in behavioral modulation,we sought for neurobiological substrates by probing its possible

plastic effects on the neuronal cytoarchitecture (Kvachnina et al.2005). Indeed, the stimulation of cultured striatal neurons with

the mixed Htr (1a/7) agonist, 8-OH-DPAT, induced a markedneurite outgrowth. These effects were specifically triggered by

Htr7 activation, because they were blocked by application of theHtr7 selective antagonist, SB-269970. Noteworthy, the applica-

tion of DA receptor agonists, well known to trigger plastic CPu/Acb changes (Geldwert et al. 2006; Gerfen 2000), did not have

any effect on the neurite length nor on other morphological

parameters in our culture conditions, thus strengthening thespecificity of the 8-OH-DPAT-mediated effects.

Our data are consistent with the increased neurite elonga-tion previously reported in mouse hippocampal neurons

following Htr7 activation (Kvachnina et al. 2005) and furthersupport the crucial role of this receptor in the modulation of

neuronal morphology. The effects observed following Htr7stimulation in vitro could be exploited in vivo to exert

beneficial effects on behavioral output. Similarly to adoles-cent MPH, a direct Htr7-targeting drug might act through

a plastic morphofunctional remodeling of the Htr7-expressingforebrain neurons, possibly in the accumbens. Further studies

are required to confirm this hypothesis, and development ofnovel Htr7 agonist drugs would certainly help.

Htr7 function and cognitive impulsivity

Present data provide evidence for a direct relationshipbetween tonic Htr7 function and the modulation of impulsive

behavior and self-control capacity. A persistent overexpres-sion of the Htr7 gene in the striatal complex, generated by

adolescent MPH administration, was indeed accompanied inthe behavioral domain by a reduced basal level of cognitive

impulsivity (see also Adriani et al. 2007). Accordingly, in thepresent study, an acute drug-induced blockade of such over-

expressed Htr7 function effectively reversed the reduction ofbasal impulsivity produced by MPH.

Self-control, a component of decision-making processes,requires: (1) to withhold the rapid ‘subcortical’ processing of

contingencies and (2) to wait for the slower elaboration ofa ‘planned’ decision, by foresight on the future consequences

of a given action or choice strategy (McClure et al. 2004;

Ridderinkhof et al. 2004). The positive modulatory effects ofMPH in decreasing impulsivity levels suggest a more efficient

(cortical) decision-making process, possibly through enhancedcorticolimbic cross-talk (Cardinal et al. 2001; Christakou et al.

2001). The observed Acb overexpression of Htr7 might beinvolved in these respects. It is worth underlining that Htr7 is

involved in ultra- and circadian rhythms, via the hypothalamicinternal clock (Hedlund & Sutcliffe 2004). Any effect in the

delay-discounting task, found in rats treated with Htr7 agonistsand/or antagonist, could reflect a difference in the internal

perception of time, rather than effects on cognitive impulsivity.Some results in behavioural tasks show that 8-OH-DPAT may

disrupt timing abilities (Chiang et al. 2000). In the present study,we decided to probe the drug challenge only at the highest

delays, so that rats were able to face the introduction andprogression of delays under fully untouched ability to evaluate

the elapsing time. The first half of all ID-task testing phaseswas thus unaffected by altered perception of the delay

intervals. As for the three drug challenge sessions, as a matterof fact, we cannot exclude that rats were detecting delay

intervals as lasting less (or more) and thus bearing them more(or less) easily. Anyway, time estimation is one of the factors,

which does account for decision making in hyperbolic delay-discounting models (Ho et al. 1999; Kheramin et al. 2003), and

the role of Htr7 in cognitive impulsivity may well tap onto delay-interval affective impact, rather than onto corticostriatal cross-

talk and top-down inhibitory control.

In addition, the selective blockade of Htr7 per se mayenhance impulsive behavior in adult rats, depending on intrinsic

and extrinsic factors such as age and/or previous handlingexperience. Recently, in vitro experiments have reported

inverse agonist properties for the SB269970 (Rauly-Lestienneet al. 2007), which may therefore explain the pro-impulsive

actions of this compound. Alternatively, serotonin is knownto exert a tonic control over intolerance-to-delay processes

(Talpos et al. 2006; van den Bergh et al. 2006; Winstanley et al.2004), with Htr7 possibly playing an essential role. However,

participation of Htr1a in the general control of impulsivebehavior cannot be ruled out. Accordingly, a combined involve-

ment of Htr7 and Htr1a has been described for regulatorymechanisms, with both receptor subtypes acting in the same

direction (Faure et al. 2006; Guscott et al. 2005; Heisler et al.1998; Landry et al. 2006). Hence, anti-impulsive properties

might be proposed for drugs acting at either receptor, althoughcomplex interactions between these receptors deserve further

investigations. Fully validated, selective drugs for Htr7 overHtr1a are not available yet, unfortunately: the most useful

compound to target Htr7 is 8-OH-DPAT, which however hasbeen used for decades as a classical Htr1a agonist. Available

literature depicts 8-OH-DPAT‘s abilities to decrease prematureresponding in reaction-time tasks, following intra-PFC infusion

(Winstanley et al. 2003) and systemic administration (Bloklandet al. 2005), but not subcutaneously (Carli & Samanin 2000). We

thus decided to more deeply investigate the possible contribu-tion of Htr7 stimulation in 8-OH-DPAT’s effects on impulsivity.

In this respect, we found a consistent ‘therapeutic’ effectfor 8-OH-DPAT, similarly to what reported by some authors

(Bizot et al. 1999; Evenden & Ryan 1999; Poulos et al. 1996)but not by others (Winstanley et al. 2005). In our hands, the

8-OH-DPAT counteracted indeed any profile of altered

Genes, Brain and Behavior (2009) 8: 356–368 365

Long-term consequences of methylphenidate during adolescence

self-control baseline (as observed during adolescence, or afternaive adults were treated with Htr7 antagonist). Interestingly,

the 8-OH-DPAT did not influence baseline impulsivity in non-naive adult rats (which indeed faced previous experimental

manipulation during adolescence). We suggest that a baselineof increased impulsivity was necessary to observe the ‘ther-

apeutic’ action of this compound. In other words, it onlyreduced an already elevated impulsivity, like in adolescent rats

or when Htr7 was blocked in naive adult rats, presently. Asa matter of fact, however, Htr7 was not the primary site of

action for 8-OH-DPAT, which acted possibly through Htr1A.Altogether, we provide clear evidence that basal levels of

impulsivity may depend on the physiological tone at Htr7, andmay also be influenced by previous experimental manipula-

tion, thus tapping onto the delicate psychobiological equilib-rium underlying self-control parameters. The possibility that

selective Htr7 agonists may be useful for therapy of impulse-control disorders still remains open. Indeed, the effect of

8-OH-DPAT should be attributed to drug‘s action on a targetother than Htr7, as was evident at least from the third of our

present experiments.

Conclusion

In summary, our data show that chronic MPH exposure duringrat adolescence produces enduring structural rearrangement of

neural pathways involved in reward-based behavior, in terms of

synaptic contacts and accumbal Htr7 upregulation. We alsoshow that Htr7 stimulation plays a key role in the regulation of

neurite outgrowth in striatal neurons, at least in vitro. Finally,we show that a selective Htr7 antagonist effectively reverses

the persistent increase in the basal self-control profile, pro-duced by adolescent MPH, and may also enhance the basal

levels of behavioral impulsivity, at least in naive adult rats.Taken together, these results suggest that Htr7 may sub-

serve the persistent changes in neuronal morphologyobserved following adolescent MPH exposure, and may play

a major role in the modulation of self-control behavior (seealso Ballatz et al. 2007). Investigation on self-control capacity

may provide insights on the efficiency of corticolimbic cross-talk, possibly via accumbal Htr7 expression, thus improving

our understanding of the molecular and structural determi-nants of impulsive behavior. New avenues can be explored to

identify more selective tools for alternative therapeutic tar-gets to treat this symptom, which is common to many

neuropsychiatric disorders (Peluso et al. 2006; Swann et al.2003 2004; Winstanley et al. 2006).

Author contributions

D.L. contributed to the experimental design and carried out

the experiments on neuronal primary cultures and the Westernblots. W.A. contributed to the experimental design, treated

the animals, carried out the behavioral experiments andperformed the statistical analyses. C.C. realized the experi-

ments on neuronal markers and morphology. G.C. realizedthe experiments on neuronal markers and morphology. E.R.

realized the behavioral experiments. E.M.M. realized thebehavioral experiments and performed the statistical analy-

ses. U.P. devised the experiments on neuronal primary

cultures. M.P. devised the experiments on neuronal markersand morphology. C.P.-C. devised the experiments and coor-

dinated the experimental work. G.L. devised the experimentsand coordinated the experimental work.

Disclosure/Conflict of interest

The author(s) declare that except for income received from

their primary employer no financial support or compensationhas been received from any individual or corporate entity over

the past 3 years for research or professional service and thereare no personal financial holdings that could be perceived as

constituting a potential conflict of interest.

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Acknowledgments

Research performed along the lines of the ‘‘ADHD-sythe’’ young-investigator projects (to WA): ‘‘under-40 call’’ from the ItalianMinistry of Health (assigned); "ERC-StG" from EU-FP7 ideas(pending). Supported by the bilateral Italy–USA Program on RareDiseases ‘X-linked or autosomal rare mental retardation syn-dromes’ (7NR1), the ERARE-EuroRETT Network (ERAR/6) (toGL); by PRIN/MIUR 2007_STRNHK_002 (to CPC); by PRIN/MIUR2007_F7AJYJ_004 (to U.dP.); by the Federation of EuropeanBiochemical Societies (FEBS) fellowship (to D.L.) and theEuropean Mind and Metabolism Association (EMMA) fellowship(to E.M.M. and E.R.). Authors wish to thank B. Duvillard (Universityof Lyon, France), L. Leone (IGB, CNR, Naples, Italy), A. De Simoneand M. R. Bianco (SUN, Naples, Italy) for their invaluable help.

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