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Behavioural Brain Research 271 (2014) 325–332

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

ellular prion protein (PrPC) modulates ethanol-induced behavioraldaptive changes in mice

aniel Riala,b,∗, Pablo Pandolfoa,c, Rafael M. Bitencourta, Fabrício A. Pamplonaa,d,arin M. Moreirae, Débora Hipolidee, Patrícia A. Dombrowski f, Claudio Da Cunhaf,oger Walzg, Rodrigo A. Cunhab,h, Reinaldo N. Takahashia, Rui D. Predigera,g

Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, BrazilCNC – Center for Neuroscience and Cell Biology, University of Coimbra, PortugalDepartamento de Neurobiologia, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, BrazilD’Or Institute for Research and Education, Rio de Janeiro, BrazilDepartamento de Psicobiologia, Universidade Federal de São Paulo, UNIFESP, São Paulo, SP, BrazilDepartamento de Farmacologia, Universidade Federal do Paraná, UFPR, Curitiba, PR, BrazilCentro de Neurociências Aplicadas (CeNAp), Hospital Universitário, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, BrazilFMUC – Faculty of Medicine, University of Coimbra, Portugal

i g h l i g h t s

Genetic deletion of PrPC decreases ethanol consumption at higher concentrations.PrPC is pivotal to rapid tolerance acquisition.D1-receptor blockade disrupts the abnormal ethanol consumption in PrPC-KO mice.PrPC is important to D1 receptor desensitization after chronic exposure to ethanol.

r t i c l e i n f o

rticle history:eceived 11 April 2014eceived in revised form 28 May 2014ccepted 31 May 2014vailable online 26 June 2014

eywords:ellular prion protein (PrPC)thanol

a b s t r a c t

Chronic consumption of drugs with addictive potential induces profound synaptic changes in the dopa-minergic mesocorticolimbic pathway that underlie the long-term behavioral alterations seen in addictedsubjects. Thus, exploring modulation systems of dopaminergic function may reveal novel targets to inter-fere with drug addiction. We recently showed that cellular prion protein (PrPC) affects the homeostasis ofthe dopaminergic system by interfering with dopamine synthesis, content, receptor density and signalingpathways in different brain areas. Here we report that the genetic deletion of PrPC modulates ethanol(EtOH)-induced behavioral alterations including the maintenance of drug seeking, voluntary consump-tion and the development of EtOH tolerance, all pivotal steps in drug addiction. Notably, these behavioral

opamineehavioronsumptionapid tolerance

changes were accompanied by a significant depletion of dopamine levels in the prefrontal cortex andreduced dopamine D1 receptors in PrPC knockout mice. Furthermore, the pharmacological blockade ofdopamine D1 receptors, but not D2 receptors, attenuated the abnormal EtOH consumption in PrPC knock-out mice. Altogether, these findings provide new evidence that the PrPC/dopamine interaction plays apivotal role in EtOH addictive properties in mice.

© 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author at: CNC – Center for Neuroscience and Cell Biology, Uni-ersity of Coimbra, Portugal. Tel.: +351 916473125; fax: +351 916473125.

E-mail address: rialdaniel@gmail.com (D. Rial).

ttp://dx.doi.org/10.1016/j.bbr.2014.05.067166-4328/© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Cellular prion protein (PrPC) is a highly conserved protein witha sparse distribution in the brain [1]. The presence of this protein

at presynaptic and postsynaptic sites [2] is coherent with the evi-dence that PrPC-null mice display altered long-term potentiation[3–5]. Although the physiological role of PrPC remains to be elu-cidated [6], neuroplasticity-related events such as aging, learning

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nd memory and brain development are affected by manipulationsf PrPC [7–9]. Considering the general agreement that neuroplas-icity plays a pivotal role in the development of drug addiction [10],t is somewhat surprising that the role of PrPC on chronic effects ofrugs with addictive potential such as ethanol (EtOH) has not been

nvestigated so far and the only study on that matter is based in ancute evaluation [11].

Chronic consumption of drugs with addictive potential, suchs EtOH, induces profound synaptic changes of the mesocorticol-mbic pathway that underlie the long-term behavioral alterationseen in addicted individuals [12]. This pathway is composed byany brain structures such as the prefrontal cortex, striatum and

ippocampus. Dopamine plays a pivotal role in such pathwaysnd, accordingly, the dopaminergic system plays a prominent rolen addiction [13]. In particular, EtOH increases the firing rate ofopaminergic neurons in these brain regions, where neuroadapta-ive structural and functional alterations related to modulation ofignaling systems, gene transcription, and protein expression takelace during addiction [14]. More recently, we showed that the Prnpenetic deletion influences dopamine synthesis, dopamine recep-ors density and intracellular signaling in different brain regions15].

The actions of dopamine are mediated through two main classesf receptor subtypes termed D1-like receptors (D1 and D5) and D2-ike receptors (D2, D3 and D4), that induce opposite intracellularesponses: D1 receptors stimulating and D2 receptors inhibitingAMP formation [16]. Both subtypes of dopamine receptors haveeen associated to rewarding, reinforcement and motivationalspects of EtOH consumption [17,18]. In particular, the blockadef dopamine D1 receptors reduces EtOH consumption [17], andncreases the reinforcing properties of EtOH in mice [19]. Moreover,tOH can modify the dopaminergic tonus by altering the num-er of dopamine transporter (DAT) expressed at the cell surface,ith a direct impact on drug-addiction process [20,21]. In keepingith the major relevance of the dopaminergic mesocorticolimbicathways for addiction, and the above-mentioned influence of PrPC

n the dopaminergic system, the aim of the present study was tonvestigate whether PrPC may impact on the development of EtOHddictive properties in mice using genetic modified mice combinedith behavioral and neurochemical assays.

. Experimental procedures

.1. Animals

Female knockout mice homozygous for a disruption of thernp gene, Prnp null mice (designated as Prnp0/0 mice), produceds previously described [22] and wild-type (Prnp+/+) mice wereonated by Dr. Vilma R. Martins (International Research Center, A.C.amargo Hospital) when they were 2–3 months old. The animalsere maintained in the animal house of the Universidade Federale Santa Catarina (Florianópolis, Brazil) until the age of 3–4 monthshen the experimental protocol started. All animals used in this

tudy weighted 20–30 g. The Prnp0/0 mice used were descendantsf Zrch I animals, while the wild-type (Prnp+/+) controls were gen-rated by crossing F1 descendants from a 129/Sv × C57BL/6J mating.he genotype of the animals was confirmed by polymerase chaineaction (PCR) with DNA extracted from the tail, using the follow-ng primers: forward (5′-ATCAGTCATCATGGCGAAC-3′) and reverse5′-AGAGAATTCTCAGCTGGATCTTCTCCCGTC-3′). A band of 693 bporresponds to the Prnp sequence in the wild-type animals, while a

and of 1635 bp represents the neomycin cassette, which replacedhe Prnp sequence and thus identifies Prnp0/0 mice. Thus, albeitrnp0/0 and Prnp+/+ mice were raised and housed side-by-side,t should be stressed that they are not littermates; therefore, the

search 271 (2014) 325–332

breeding scheme used in this study does not allow excluding theselection of compensatory mechanisms.

All rodents were kept four to a cage and were subjected to a 12-hlight/dark cycle (lights on at 7:00 a.m.) with free access to food andwater. All procedures used in the present study were approved bythe local Ethics Committee on the Use of Animals (CEUA/UFSC PP00452), which follows the NIH publication “Principles of LaboratoryAnimal Care”.

2.2. Locomotor activity

The effects of EtOH on locomotor activity of mice were inves-tigated in the open field test. The open field apparatus was a50 cm × 50 cm × 50 cm chamber illuminated to 12 lx. Before the testall animals were weighted. For testing, mice were placed in thecenter of the open field and their activity recorded during 10 min.The total distance was automatically quantified by the Any-maze®

video-tracking system (Stoelting Inc., Kiel, WI, USA). Mice wereinjected by intraperitoneal (i.p.) route with EtOH (2 g/kg) (analyt-ical grade, VETEC, RJ, Brazil) 10 min before the test during 1, 7, 14and 21 consecutive days.

2.3. Blood EtOH assay

Ten minutes after the EtOH (2 g/kg, i.p.) administration, bloodsamples were collected from the animals by direct tail puncture.Blood EtOH concentration was evaluated enzymatically based onEtOH conversion to acetaldehyde by the action of alcohol dehydro-genase [23].

2.4. EtOH voluntary consumption

All mice were isolated and exposed to two inverted bottles withmetal sippers placed on stainless steel cage tops during 24 h. Foodwas distributed near both bottles to avoid food-associated tubepreferences. One bottle always contained tap water and the othercontained increasing concentration of EtOH (3%, 6%, 10% and 20%v/v in tap water). Mice were exposed to each EtOH concentrationfor a block of 5 days, according to a previously described proce-dure [20]. The position of the two bottles was reversed daily duringthe testing period to prevent position bias. Bottles were weighteddaily and EtOH consumption was expressed as grams of EtOH perkilogram of body weight.

2.5. EtOH voluntary consumption (5 h protocol)

Mice were isolated and offered tap water and a 10% (v/v in tapwater) EtOH solution in a two-tube free-choice paradigm, using alimited-access model. Fluid intake was monitored during the test-ing period of 5 h per day during 5 consecutive days to provide anindex of consumption by weighting the bottles at the end of the 5 hperiod [17]. EtOH consumption was expressed as grams of EtOH perkg of body weight for the absolute amount consumed over the 5 htesting period. The position of the two bottles was reversed dailyduring the testing period to prevent position bias. To test the impactof dopamine receptors, the mice were treated 30 min before the testwith the dopamine D1 receptor antagonist (SCH-23390, 0.1 mg/kg,i.p.) or the dopamine D2 receptor antagonist (sulpiride, 80 mg/kg,i.p.).

2.6. Rapid tolerance to EtOH-induced motor impairment

The rapid tolerance to EtOH was measured as previouslydescribed [24] utilizing the rotarod apparatus (Rotamex-V-EE/85)controlled by a computer system (Columbus Instruments Com-puter; Columbus, OH, USA). The mice were trained under

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ontinuous acceleration (1 rpm/s) in 1-min sessions. Whenever aouse dropped off the rotating bar, it received a footshock (0.5 mA,

s). The speed at which a mouse dropped off the rotating bar wasaken as the performance score. Mice that did not reach a stableaseline (at least 20 rpm) in 10 trials were not considered for anal-sis, which represented less than 10% of mice. Prnp+/+ and Prnp0/0

ice were further divided in subgroups in order to be i.p. injectedith EtOH (1.75 g/kg) or saline. Two hours after saline or EtOH

njections, the mice received an additional dose of saline or EtOH1.0 g/kg, i.p.), in order to complete 2.75 g/kg. The procedure ofdministering EtOH in two doses (1.75 plus 1.0 g/kg) was employedecause previous experiments showed that a total dose of 2.75 g/kgn day 1 is insufficient to produce a reliable rapid tolerance onay 2 [25]. Mice then returned to their home cages. After 24 h,ll mice including controls received EtOH (1.75 g/kg, i.p) and werehen re-submitted to the rotarod test to evaluate rapid tolerancecquisition.

.7. Conditioned place preference (CPP)

The rewarding effects of EtOH consumption were evaluatedtilizing a biased CPP protocol [19]. CPP was evaluated in four iden-ical rectangular acrylic boxes. Each of the CPP boxes consisted ofhree different compartments separated by guillotine doors. Thewo conditioning compartments (23 cm × 16 cm × 15 cm) had dif-erent tactile and visual cues: one compartment was black with amooth floor and the other was black with vertical white stripesnd aluminium mesh floor. The central “neutral” compartment10 cm × 16 cm × 15 cm) was gray with a smooth floor and hadpenings (5 cm × 5 cm) that gave access to the two other compart-ents. The test was conducted under low-light conditions (10 lx).

he behavior of each mouse was recorded via a video cameraositioned above the boxes and monitored in an adjacent roomia a closed circuit TV camera. The experimenter was unawaref the drug treatment or the genotypes during behavioral evalu-tion. The apparatus was cleaned with a 70% EtOH solution andhen dried with a paper towel after each trial. The CPP protocolonsisted of a schedule of 8 days divided into three different phases:re-conditioning, conditioning, and post-conditioning. In the pre-onditioning phase (day 1), mice were allowed to freely explorehe three compartments for 15 min. The time spent by the ani-

al (with all four paws) in each of the three compartments wasecorded.

The conditioning phase (days 2–7) consisted of six 25-min ses-ions, one per day. Immediately after administration of EtOH (0.5 or.0 g/kg, i.p.), the mice were confined in one compartment and, onlternate days, received saline and were confined to the oppositeompartment. The control group received saline before condition-ng in each compartment. In the post-conditioning phase (day 8),ach animal was placed in the neutral compartment and had freeccess to all three compartments. The time spent in each compart-ent was recorded for 15 min.

.8. Measurements of monoamine levels by high performanceiquid chromatography (HPLC)

We used a previously validated HPLC separation methodo determine the levels of DA and its metabolites, 3,4-ihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA),s well as noradrenaline and serotonine [26]. Briefly, mice wereilled by decapitation, their brains were removed immediatelynd the structures of interest (prefrontal cotex and striatum) were

issected, frozen in liquid nitrogen and stored at −70 ◦C. ThePLC system consisted a LC-20AT pump (Shimadzu, São Paulo,razil) equipped with a manual Rheodyne 7725 injector with

20 �L loop feading a Synergi Fusion-RP C-18 reverse-phase

search 271 (2014) 325–332 327

column (150 mm × 4.6 mm i.d. with 4 �m particle size) protectedby a 4 mm × 3.0 mm pre-column (SecurityGuard Cartridges Fusion-RP), both maintained inside a temperature-controlled oven (25 ◦C;Shimadzu). The monoamines were identified with an electro-chemical detector (ESA Coulochem III Electrochemical Detector)equipped with a guard cell (ESA 5020) with the electrode set at350 mV and a dual electrode analytical cell (ESA 5011A) with twochambers in series: each chamber includes a porous graphite colori-metric electrode, a double counter electrode and a double referenceelectrode. Oxidizing potentials were set at 100 mV for the first elec-trode and at 450 mV for the second electrode. The tissue sampleswere homogenized with an ultrasonic cell disrupter (Sonics) in0.1 M perchloric acid containing 0.02% sodium metabisulfite andan internal standard. After centrifugation at 10,000 g for 30 min at4 ◦C, 20 �L of the supernatant was injected into the chromatograph.The mobile phase, used at a flow rate of 1 mL/min, had the follow-ing composition: 20 g citric acid monohydrated (Merck, Darmstadt,Germany), 200 mg octane-1-sulfonic acid sodium salt (Merck),40 mg EDTA (Sigma), in 10% (v/v) methanol (Merck, Darmstadt,Germany) to a final volume of 1000 mL with HPLC-grade water. ThepH was adjusted to 4.0 and the mobile phase was filtered througha 0.45 �m filter before use. The peak areas of the external stan-dards were used to quantify the metabolites of interest in the testedsamples.

2.9. Autoradiography

Prnp+/+ and Prnp0/0 mice were sacrificed by decapitation andtheir brains were immediately removed, frozen over dry ice andstored at −80 ◦C until cryostat sectioning. Serial 20 �m coro-nal sections were cut on Leica cryostat at −20 ◦C, collected ontoglass slides and stored at −80 ◦C until the day of the assays.Dopamine D1 receptor binding was probed with [3H]-SCH-23390,dopamine D2 receptor binding was detected using [3H]-raclopride,and the density of DAT was evaluated with [3H]-WIN-35248.Briefly, the sections were brought to room temperature and thenpre-incubated in 50 mM Tris buffer (pH 7.4) containing 120 mMNaCl, 4 mM MgCl2, 1 mM EDTA and 1.5 mM CaCl2, for 30 (D1receptor and DAT) or 15 (D2 receptor) min. Sections were thenincubated in Tris buffer containing either 2 nM [3H]-SCH-23390(86.0 Ci/mmol, Perkin Elmer Life Science, Boston, USA) for 90 minat 37 ◦C (D1 receptor), 2 nM [3H]-raclopride (76.0 Ci/mmol, PerkinElmer Life Science, Boston, USA) for 2 h at room temperature (D2receptor) or 10 nM [3H]-WIN 35248 (85.0 Ci/mmol; Perkin-ElmerLife Science, Boston, USA) for 2 h at room temperature (DAT). Sec-tions were then washed once (D1 receptor) or twice (D2 receptorand DAT) in ice-cold Tris buffer for 5 min, followed by one quick diprinse of ice-cold distilled water before drying. Slices were exposedto Kodak Biomax MR-1 in tungsten cassettes together with cali-brated standards for 4 weeks. All groups were represented in eachfilm. Films were developed and densitometry analyses were per-formed using the MCID system (Imaging Research, St. Catherine’s,Ontario, Canada). At least five sections were measured to obtainvalues for each mouse for each region. Anatomical regions weredefined according to the mouse brain atlas of [27].

2.10. Statistical analysis

All data are expressed as mean ± s.e.m. and the statistical anal-ysis was carried out using two- or three-way analysis of variance(ANOVA) for multifactorial comparison, provided that they passedthe Shapiro–Wilk’s W normality test. ANOVA with repeated mea-

sure was used for comparisons of subsequent dependent groups(usually repeated treatment). Genotype, treatment and time wereused as independent variables in the statistical analysis. Fol-lowing significant ANOVAs, multiple post hoc comparisons were

328 D. Rial et al. / Behavioural Brain Research 271 (2014) 325–332

Fig. 1. Effects of PrPC genetic deletion on the EtOH-induced locomotor activity.(a) Distance traveled (mean ± s.e.m.) by Prnp+/+ and Prnp0/0 mice after a single i.p.injection of saline or EtOH (2 g/kg). *P < 0.05 compared to Prnp0/0 saline, #P < 0.05compared to EtOH-treated Prnp+/+ group using a Newman–Keuls post hoc test;n = 9–10. (b) Distance traveled (mean ± s.e.m.) by Prnp+/+ and Prnp0/0 mice after7*t

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Table 1Effects of Prnp genetic ablation and ethanol treatment on mice body weight.

Genotype Treatment Day 1 Day 7 Day 14 Day 21

Prnp+/+ Saline 23.4 ± 0.33 22.0 ± 0.39 22.2 ± 0.32 21.1 ± 0.43

TE

, 14 and 21 sequential i.p. injections of saline or EtOH (2 g/kg) (mean ± s.e.m.).P < 0.05 compared to saline-treated Prnp0/0 group, #P < 0.05 compared to EtOH-reated Prnp+/+ group using a Newman–Keuls post hoc test; n = 9–10.

erformed using the Newman–Keuls test. The accepted level of sig-ificance for the tests was P ≤ 0.05. All tests were performed usinghe Statistica software package (StatSoft Inc., Tulsa, OK, USA).

. Results

.1. Genetic deletion of PrPC alters the EtOH effects on locomotorctivity

Acute administration of EtOH (2 g/kg, i.p.) increased locomo-ion in Prnp0/0 but not in Prnp+/+ mice (Fig. 1a) [(genotype factor:F1,35) = 11.3, P < 0.05), (treatment factor: (F1,35) = 17.4, P < 0.05),genotype × treatment: (F1,35) = 1.4, P > 0.05)]. The same pattern ofocomotor alteration was observed after 7, 14 and 21 days of con-ecutive EtOH administration (2 g/kg, i.p.) (Fig. 1b) [(Day 7 genotypeactor: (F1,36) = 10.7, P < 0.05), (Day 7 treatment factor: (F1,36) = 11.8,

< 0.05), (Day 7 genotype × treatment: (F1,36) = 3.6, P > 0.05), (Day4 genotype factor: (F1,36) = 2.5, P > 0.05), (Day 14 treatmentactor: (F1,36) = 21.4, P < 0.05), (Day 14 genotype × treatment:F1,36) = 3.1, P > 0.05), (Day 21 genotype factor: (F1,36) = 4.8, P < 0.05),Day 21 treatment factor: (F1,36) = 12.3, P < 0.05), (Day 21 geno-ype × treatment: (F1,36) = 2.3, P > 0.05)]. No significant inter-strain

ifferences on the body weight and blood EtOH levels during the 21ays of EtOH administration were observed (Tables 1 and 2 respec-ively). Additionally, after 21 days of EtOH administration, hepaticistology indicated that Prnp+/+ and Prnp0/0 mice did not present

able 2ffects of ethanol treatment on blood ethanol concentration of Prnp+/+ and Prnp0/0 mice.

Genotype Treatment Day 1

Prnp+/+ EtOH 106.0 ± 0.91

Prnp0/0 EtOH 115.0 ± 0.91

EtOH 23.1 ± 0.76 21.5 ± 0.74 21.9 ± 0.70 22.3 ± 0.71

Prnp0/0 Saline 23.3 ± 0.76 21.8 ± 0.64 22.5 ± 0.67 22.9 ± 0.64EtOH 23.9 ± 0.52 21.8 ± 0.53 21.6 ± 0.37 22.0 ± 0.42

any signs of steatosis, cirrhosis or evident lymphocytic infiltrates(data not shown).

3.2. Genetic deletion of PrPC prevents escalating EtOH intake inmice: role of dopamine receptors

In a voluntary consumption protocol with increasing con-centrations of EtOH, no significant inter-strain differences inthe intake of 3% or 6% EtOH solutions were observed. How-ever, Prnp0/0 mice drunk significantly less of the solutions withhigher EtOH concentrations (10% and 20%) compared to Prnp+/+

mice (Fig. 2a) [(genotype factor: (F1,17) = 6.0, P < 0.05), (repetitionfactor: (F3,51) = 19.7, P < 0.05), (genotype × repetition: (F3,51) = 1.5,P > 0.05)]. To investigate the participation of dopamine D1 andD2 receptors in the observed phenotype of Prnp0/0 mice, thedopamine receptor antagonists SCH-23390 (0.1 mg/kg, i.p.) orsulpiride (80 mg/kg, i.p.) were injected daily in the same self-administration protocol with 10% EtOH solution. Surprisingly,Prnp0/0 mice actually consumed more EtOH than controls atDay 1 of the self-administration protocol. Pretreatment withthe dopamine D1 receptor antagonist (SCH-23390), but not withthe dopamine D2 receptor antagonist (sulpiride), suppressedthis initial increased consumption of EtOH by Prnp0/0 mice(Fig. 2b). Over the following days, the pattern of EtOH intakewas swapped between the two strains: at day 5, Prnp0/0 miceexhibited decreased EtOH intake in comparison with Prnp+/+ mice,which exhibited escalating consumption of EtOH across days.Blockade of dopamine D1 or D2 receptors also disrupted theescalating consumption of EtOH in Prnp+/+ mice (Fig. 2b) [(geno-type factor: (F1,28) = 0.8, P > 0.05), (treatment factor: (F2,28) = 3.7,P < 0.05), (genotype × treatment: (F2,28) = 1.3, P > 0.05), (repetitionfactor: (F4,112) = 4.2, P < 0.05), (repetition × genotype: (F4,112) = 6.3,P < 0.05), (repetition × treatment: (F8,112) = 3.8, P < 0.05) and (repe-tition × genotype × treatment: (F8,112) = 4.5, P < 0.05)].

3.3. Genetic deletion of PrPC disrupts the development of rapidtolerance and increases the susceptibility to rewarding effects ofEtOH

Tolerance to the behavioral effects of EtOH is a potential indi-cator of the future development of alcoholism [28]. Therefore, weused a 2-days repeated administration protocol of rapid toleranceto EtOH in the rotarod apparatus to investigate whether PrPC dele-tion would interfere with the plasticity necessary to adapt to EtOHeffects. Prnp+/+ mice developed rapid tolerance to EtOH-inducedmotor incoordination, while Prnp0/0 mice did not (Fig. 2c) [(geno-type factor: (F1,24) = 45.0, P < 0.05), (treatment factor: (F1,24) = 306.3,

P < 0.05), (genotype × treatment: (F1,24) = 7.6, P < 0.05), (repetitionfactor: (F1,24) = 238.8, P < 0.05), (repetition × genotype: (F1,24) = 9.7,P < 0.05), (repetition × treatment: (F1,24) = 314.4, P < 0.05) and (rep-etition × genotype × treatment: (F1,24) = 33.8, P < 0.05)]. This may

Day 7 Day 14 Day 21

114.0 ± 0.91 105.0 ± 0.91 111.0 ± 0.97105.0 ± 0.91 124.0 ± 0.91 127.0 ± 1.7

D. Rial et al. / Behavioural Brain Research 271 (2014) 325–332 329

Fig. 2. Effects of PrPC genetic deletion on EtOH voluntary consumption and rewarding properties. (a) EtOH voluntary consumption (g/kg/day) (mean ± s.e.m.) of Prnp+/+ andPrnp0/0 mice exposed to increasing EtOH concentrations. *P < 0.05 compared to Prnp+/+ mice; n = 9–10. (b) 10% EtOH solution voluntary consumption (g/kg/5 h) (mean ± s.e.m.)of Prnp+/+ and Prnp0/0 mice daily pretreated with D1 (SCH-23390) and D2 receptor antagonists (sulpiride). *P < 0.05 compared to EtOH consumption on Day 1 of the respectivegenotype. #P < 0.05 compared to the performance of saline-treated Prnp+/+ mice in day 1; &P < 0.05 compared to saline-treated mice of the same genotype and on the sameday, both using a Newman–Keuls post hoc test; n = 8–10. (c) Latency to fall(s) (mean ± s.e.m.) of Prnp+/+ and Prnp0/0 mice submitted to the rapid tolerance task. *P < 0.05c EtOHn or EtOC eatme

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ompared to saline-treated mice of the respective genotype, #P < 0.05 compared to = 9–10. (d) Time (s) (mean ± s.e.m.) spent by Prnp+/+ and Prnp0/0 mice after salinePP. *P < 0.05 compared to preconditioning phase of the respective genotype and tr

e suggestive of an increased susceptibility to EtOH effects overime. Consistent with this view, in the conditioned place preferenceCPP) test, the lowest dose of EtOH (0.5 g/kg, i.p.) induced CPP in thernp0/0 group only, while a 4 times higher dose of EtOH (2.0 g/kg,.p.) was required to induce CPP in Prnp+/+ mice (Fig. 2d) [(geno-ype factor: (F1,30) = 0.1, P > 0.05), (treatment factor: (F2,30) = 2.1,

> 0.05), (genotype × treatment: (F2,30) = 3.1, P > 0.05), (repetitionactor: (F1,30) = 8.1, P < 0.05), (repetition × genotype: (F1,30) = 0.008,

> 0.05), (repetition × treatment: (F2,30) = 2.3, P > 0.05) and (rep-tition × genotype × treatment: (F2,30) = 3.7, P < 0.05)]. Altogether,hese data suggest a higher sensitivity of Prnp0/0 mice to EtOH-nduced reward, with reduced capacity to tolerate its effects overhe time.

.4. PrPC modulates dopaminergic system plasticity followinghronic EtOH administration

In view of the key involvement of the dopaminergic systemn the impact of PrPC on EtOH intake, we next compared thehanges of the dopaminergic system caused by chronic EtOHdministration in Prnp0/0 and Prnp+/+ mice. The dopamine levels

n the prefrontal cortex and in the striatum after 21 consecu-ive days of saline or EtOH (2 g/kg, i.p.) treatment are depictedn Fig. 3a and b. EtOH treatment did not alter dopamine lev-ls in the prefrontal cortex of Prnp+/+ mice, but significantly

-treated Prnp0/0 on Day 1 and Day 2 (EE), using the Newman–Keuls post hoc test;H (0.5 or 2 mg/kg) paired-side in preconditioning and postconditioning phases of

nt; n = 9–10.

increased dopamine levels in the Prnp0/0 mice [(genotype fac-tor: (F1,16) = 0.1, P > 0.05), (treatment factor: (F1,16) = 0.8, P > 0.05)and (genotype × treatment: (F1,16) = 4.9, P < 0.05)]. We did notobserve any alteration concerning treatment or genotype in thestriatal dopamine levels [(genotype factor: (F1,16) = 0.01, P > 0.05)(treatment factor: (F1,16) = 0.2, P > 0.05), (genotype × treatment:(F1,16) = 0.4, P > 0.05)]. In accordance with previous results [29],repeated EtOH treatment decreased dopamine D1 receptor den-sity in wild-type mice in all striatal sub-regions analyzed (Fig. 4),namely in the dorso-medial caudate putamen (DMCPu) [genotypefactor: (F1,10) = 3.9, P > 0.05; treatment factor: (F1,10) = 0.1, P > 0.05;genotype × treatment: (F1,10) = 8.1, P < 0.05] (Fig. 4b), ventro-lateralcaudate putamen (VLCPu) [genotype factor: (F1,10) = 1.8, P > 0.05;treatment factor: (F1,10) = 0.7, P > 0.05; genotype × treatment:(F1,10) = 12.2, P < 0.05] (Fig. 4c) and nucleus accumbens core (AccCore) [genotype factor: (F1,10) = 2.8, P > 0.05; treatment factor:(F1,10) = 0.6, P > 0.05; genotype × treatment: (F1,10) = 6.4, P < 0.05](Fig. 4d). Remarkably, the same treatment did not modify D1receptor density in the striatum of Prnp0/0 mice. Fig. 4a showsa representative image of D1 receptor density in the striatumof both mouse strains treated during 21 days with saline or

EtOH (2 g/kg). The dopamine D2 receptors and DAT densitiesin both striatum and the prefrontal cortex were not signifi-cantly altered regardless of the genotype or treatment (data notshown).

330 D. Rial et al. / Behavioural Brain Research 271 (2014) 325–332

F e levea . #P < 0

4

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FP(#

ig. 3. Impact of PrPC genetic deletion on the EtOH-induced alterations of dopaminnd Prnp0/0 mice. *P < 0.05 compared to saline-treated mice with the same genotype

. Discussion

The role of PrPC in synaptic plasticity is well described [3,30].hus, it is reasonable to hypothesize that PrPC could influencehe establishment of drug addiction, as functional and structurallterations are observed in the addicted brain. The present studyrovides the first evidence that the genetic manipulation of PrPC

nterferes with EtOH effects in mice through alterations in theopaminergic system. Considering the pivotal role of the dopa-inergic system in drug addiction and that we recently described

hat PrPC modulates several features of dopaminergic system [15],

ere we hypothesized that PrPC deletion may affect the behav-

oral adaptive changes following repeated EtOH administration inice.

ig. 4. Impact of PrPC genetic deletion on the EtOH-induced alterations of D1 receptor

rnp+/+ and Prnp0/0 mice treated with saline or EtOH. (b) Autoradiographic quantification

DMCPu), (c) ventrolateral caudate putamen (VLCPu) and (d) nucleus accumbens core

P < 0.05 compared to saline-treated Prnp+/+, using a Newman–Keuls post hoc test; n = 5.

ls. (a) DA levels (mean ± s.e.m.) in the prefrontal cortex (a) and striatum of Prnp+/+

.05 compared to saline-treated Prnp+/+, using a Newman–Keuls post hoc test; n = 5.

The effects of EtOH on locomotor activity in rodents also occurvia modifications of dopaminergic function [31]. Either acute orchronic EtOH treatment increased locomotion in Prnp0/0 mice, sug-gesting that PrPC attenuates the euphoric/hyperlocomotor effectsof EtOH. This increased locomotion in EtOH-treated Prnp0/0 micewas maintained after 21 days of repeated EtOH administration. Pos-sible differences in EtOH metabolism in Prnp0/0 mice are unlikelyexplanations for this result, since the blood levels of EtOH were sim-ilar between mouse strains and hepatic histological lesions wereabsent in chronically treated mice of both groups. Altogether, theseresults raise two important points: first, there is an acute influence

of PrPC on EtOH effects that transcends possible developmentalalterations observed in PrPC-null mice; and second, there mightbe a direct impact of PrPC modulation on EtOH effects, in addition

density. (a) Representative dopamine D1 receptor autoradiography of the STR of(�Ci/g) (mean ± s.e.m.) of D1 receptor density in the dorsomedial caudate putamen(Acc Core). *P < 0.05 compared to saline-treated mice of the respective genotype.

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D. Rial et al. / Behavioural Br

o the likely influence of dopaminergic alteration, as observed inrnp0/0 mice [15].

Following these initial findings, we submitted Prnp+/+ andrnp0/0 mice to a rapid tolerance protocol aiming to investigate pos-ible strain differences in the short-term neuroadaptation to EtOH.mportantly, rapid tolerance to EtOH contributes to voluntary EtOHonsumption and is indicative of an increased susceptibility to alco-olism [32]. Genetic ablation of PrPC prevented the developmentf rapid tolerance to EtOH-induced motor incoordination in miceested in the rotarod. Therefore, in the absence of PrPC protein, the

agnitude of the central effects of EtOH are sustained for prolongederiods after repeated administration [10].

The absence of rapid tolerance to EtOH also interferes witholuntary consumption. Successful short-term adaptation to EtOHrompts a subsequent abnormally higher EtOH consumption, typ-

cal of alcoholism in humans. This was confirmed by testing thetOH oral-self administration with increasing concentrations oftOH. When testing lower concentrations of EtOH solution (3–6%),oth strains consumed similar amounts of EtOH; however, Prnp0/0

ice consumed less of the 10% and 20% EtOH solutions in compar-son with Prnp+/+ mice. Interestingly, when the intake profile of a0% EtOH solution was evaluated day-by-day, Prnp0/0 mice actu-lly consumed more EtOH than Prnp+/+ mice in the first day, buthis pattern was inversed over the days. This observation indicateshat: (1) the altered dopaminergic system of Prnp0/0 mice confers aigher sensitivity to the initial EtOH exposure (day 1); and (2) the

ack of appropriate adaptation to the effects of EtOH (e.g., lack oftOH tolerance) may lead these animals to reduce EtOH consump-ion over the following days. The key involvement of modificationsf the dopaminergic system in this PrPC-mediated control of EtOHonsumption was highlighted by the different impact of the sys-emic administration of selective dopamine D1 and D2 receptorntagonists on the EtOH consumption in PrPC-null when comparedo control mice. Thus, the pretreatment with either dopamine D1r D2 receptor antagonists blocked the escalating pattern of EtOHonsumption in Prnp+/+ mice, while keeping normal “acute” intaken day 1. On the other hand, only D1 receptor antagonism alteredhe profile of EtOH consumption (especially on day 1), indicatinghe fundamental role of this receptor in the altered acute responseso EtOH in Prnp0/0 mice.

The neurochemical adaptive changes of dopamine metabolismnd receptors in response to repeated EtOH treatment were furtherxplored. Following 21 days of EtOH administration, the dopamineevels were selectively increased in the prefrontal cortex of Prnp0/0,

hereas no significant changes in striatal dopamine levels wereound regardless the genotype. The lack of alteration in dopamineevels of Prnp+/+ mice was expected due to the ability of mice todapt to chronic EtOH exposure, i.e., reaching an allostatic state14]. On the other hand, and as already observed in the rapidolerance experiments, PrPC-null mice were unable to activate

echanisms of neuronal plasticity and adapt to repeated EtOHxposure, with the consequence that they did not reach an allostaticalance, at least during the current period of analysis (21 days).his observation is in line with the current autoradiographic results.fter 21 days of consecutive EtOH treatment, the Prnp+/+ group dis-layed a reduced dopamine D1 receptor density in the striatum, buthe density of D1 receptor remained similar to the saline-treatedroup in Prnp0/0 mice. Altogether, these findings demonstrate thathe adaptations of the dopaminergic system caused by EtOH arerucially dependent on the presence of PrPC.

As the drug seeking is a key element in EtOH addiction [19],e evaluated Prnp+/+ and Prnp0/0 in the CPP task. Confirming the

pen field findings of increased sensitivity of Prnp0/0 mice to EtOH,hese animals developed CPP at a lower EtOH dose (0.5 g/kg/i.p),hereas Prnp+/+ mice developed CPP only at the highest EtOH dose

ested (2 g/kg/i.p). These results reinforce the altered impact of

search 271 (2014) 325–332 331

EtOH in Prnp0/0 mice. The prefrontal cortex and striatum were pre-viously described as important brain regions for the CPP acquisition[19], and we reported recently that Prnp0/0 mice display dopami-nergic modifications in these two brain regions [15]. We suggestthat the alteration of the range of the biphasic effects of EtOH(euphoric/dysphoric) might be fundamentally dependent of dopa-minergic alterations in prefrontal cortex and striatum, which areunder control of PrPC.

This novel dopaminergic-based hypothesis to interpret theimpact of PrPC on the central effects of EtOH does not exclude thepossibility that PrPC might also control the effects of EtOH on glu-tamatergic synapses, as recently proposed to occur in hippocampalsynapses [11]. Indeed, the sensitization to psychoactive drugs isincreasingly recognized to result from adaptive changes in the plas-ticity of glutamatergic synapses, but mainly of the striatum ratherthan of the hippocampus [33].

5. Conclusion

In summary, we here reported that PrPC is involved in the EtOH-induced neurobehavioral adaptive changes in mice and that geneticmanipulation of this protein alters several indicators of EtOH addic-tion. It is important to highlight that the entire study was carriedout comparing Prnp0/0 and Prnp+/+ raised in parallel rather thanusing littermates, which limits the strength of the observationsin spite of their remarkable coherency. Thus, we systematicallyobserved a lack of behavioral adaptation to EtOH observed in PrPC-null mice, which underscores the importance of this protein inneuroplasticity. Evidence obtained in a previous study suggests thatPrPC influences the balance of the dopaminergic transmission insome brain areas [15], probably directly related to the resilienceagainst EtOH effects and allowing the individual to cope with EtOHeffects to a given extent. Taken together, the results of the presentstudy indicate that PrPC is fundamental for the physiology of themesocorticolimbic dopaminergic transmission, driving adaptivefluctuations in the levels of dopamine D1 receptors. This suggeststhat manipulation of PrPC may be a novel potential therapeuticstrategy to manage alcoholism. How this manipulation affects othereffects of potentially addictive drugs is still an open question thatcertainly deserves future investigation.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by grants from the following Brazil-ian institutions: Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq, Brazil), Coordenacão de Aperfeic oamento dePessoal de Nível Superior (CAPES, Brazil), Programa de Apoio aosNúcleos de Excelência (PRONEX-NENASC Project), Fundac ão deApoio à Pesquisa do Estado de Santa Catarina (FAPESC), Fundac ãopara a Ciência e Tecnologia (PTDC/SAU-NMC/114810/2009,Portugal) and CAPES/FCT. D.R. received scholarships from CNPq.R.W., R.N.T. and R.D.P. are supported by research fellowships fromCNPq, Brazil.

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