Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats

Intermittent ethanol exposure induces inflammatory braindamage and causes long-term behavioural alterations inadolescent rats

Maria Pascual,1 Ana M. Blanco,1 Omar Cauli,2 Jose Minarro3 and Consuelo Guerri11Department of Cellular Pathology, Centro de Investigacion Prıncipe Felipe, Avda. Autopista del Saler, 16. 46013-Valencia, Spain2Department of Neurobiology, Centro de Investigacion Prıncipe Felipe, Avda. Autopista del Saler, 16. 46013-Valencia, Spain3Department of Psychobiology, Facultad de Psicologıa, Universitat de Valencia, Avda. Blasco Ibanez, 21. 46010-Valencia, Spain

Keywords: adolescence, indomethacin, inflammation, intermittent ethanol intoxication, neurobehaviour

Abstract

Adolescent brain development seems to be important for the maturation of brain structures and behaviour. Intermittent binge ethanoldrinking is common among adolescents, and this type of drinking can induce brain damage. Because we have demonstrated thatchronic ethanol treatment induces inflammatory processes in the brain, we investigate whether intermittent ethanol intoxicationenhances cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in adolescent rats, and whether these mediatorsinduce brain damage and cause permanent cognitive dysfunctions. Adolescent rats were exposed to ethanol (3.0 g ⁄ kg) for twoconsecutive days at 48-h intervals over 14 days. Levels of COX-2, iNOS and cell death were assessed in the neocortex,hippocampus and cerebellum 24 h after the final ethanol administration. The following day or 20 days after the final injection (adultstage), animals were tested for different behavioural tests (conditional discrimination learning, rotarod, object recognition, beam-walking performance) to assess cognitive and motor functions. Our results show that intermittent ethanol intoxication upregulatesCOX-2 and iNOS levels, and increases cell death in the neocortex, hippocampus and cerebellum. Furthermore, animals treated withethanol during adolescence exhibited behavioural deficits that were evident at the end of ethanol treatments and at the adult stage.Administration of indomethacin, a COX-2 inhibitor, abolishes the induction of COX-2 and iNOS expression and cell death, preventingethanol-induced behavioural deficits. These findings indicate that binge pattern exposure to ethanol during adolescence inducesbrain damage by inflammatory processes and causes long-lasting neurobehavioural consequences. Accordingly, administeringindomethacin protects against ethanol-induced brain damage and prevents detrimental ethanol effects on cognitive and motorprocesses.

Introduction

The brain is one of the major target organs for ethanol actions, andheavy alcohol consumption results in significant alterations of thebrain structure, physiology and function (Harper & Matsumoto, 2005).Recent studies showed that episodic alcohol intoxication, or binge-type drinking, in experimental animals can also result in both braininjury and neurodegeneration in corticolimbic areas (Crews et al.,2004), which are involved in many aspects of learning and spatialmemory (Haberly & Bower, 1989).

Intermittent ethanol binging is common during adolescence. Incomparison with adult drinking, however, relatively little is knownabout adolescent drinking and its prolonged neurobiological impactduring this developmental period (Monti et al., 2005). Duringadolescence, the brain undergoes neuromaturation, which involveschanges in neurotransmission and plasticity that are associated withstructural modifications in some brain regions, including the hippo-campus, prefrontal cortex and the limbic system structures (Dahl,2004). Indeed, clinical and experimental studies have provided

evidence of the special sensitivity of the adolescent brain to someeffects of ethanol, such as memory impairments (White & Swartz-welder, 2005) and ethanol-induced brain damage (Crews et al., 2000).The underlying mechanisms of ethanol-induced brain damage

during binge drinking are poorly understood, although some reportssuggest the participation of excitotoxic events (Prendergast et al.,2004).We have recently reported that chronic ethanol intake inducesinflammatory mediators in the brain by activating glial cells andstimulating intracellular signalling pathways that trigger induction ofcytokines, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase(iNOS) and neural cell death (Valles et al., 2004; Blanco et al., 2005).Notably, the glial activation associated with upregulation of inflam-matory molecules plays an important role in neuron loss in someneurodegenerative diseases (Campbell, 2004). In addition, elevatedlevels of COX-2 and iNOS are observed during excitotoxicity,ischaemia and neural injury (Heales et al., 1999; O’Banion, 1999;Yamada et al., 1999). These changes are associated with neurobe-havioural and cognitive deficits (Andreasson et al., 2001; Li et al.,2004).COX-2 is induced in several cell types in response to damage and

inflammatory molecules, and upregulation of the COX-2 expression hasbeen described in the brain after traumatic injury and chronic

Correspondence: Dr C. Guerri, as above.E-mail: [email protected]

Received 2 August 2006, revised 26 October 2006, accepted 15 November 2006

European Journal of Neuroscience, Vol. 25, pp. 541–550, 2007 doi:10.1111/j.1460-9568.2006.05298.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

degenerative disorders (Knott et al., 2000). Accordingly, COX-2inhibitors prevent neuronal loss and ameliorate brain injury caused byexcitotoxicity (Iadecola et al., 2001), ischaemic brain injury, and alsobrain inflammation and neurodegeneration (Willard et al., 2000;Minghetti, 2004). Recent reports suggest that non-steroidal anti-inflammatory drugs (NSAIDs), which include COX-2 inhibitors, reducethe risk of developing Alzheimer’s disease (Andreasson et al., 2001),and improve behavioural and cognitive functions (Minghetti, 2004).Based on the above observations and the vulnerability of the

adolescent brain to deleterious effects of ethanol, we hypothesize thatintermittent ethanol intoxication during adolescence induces braindamage by inflammatory processes, impairs the normal brain matur-ation and plasticity, and causes long-lasting behavioural consequences.To further support this hypothesis, we assessed whether the admin-istration of a NSAID, such as indomethacin, protects ethanol-inducedbrain damage and prevents the detrimental effects of ethanol oncognitive and motor processes.

Materials and methods

Animals and treatments

Ninety Wistar rat pups (Harlan, Barcelona, Spain) were used assubjects in these experiments. Pups remained housed with respectivedams in a temperature- and humidity-controlled vivarium in a 12 hlight : dark cycle until postnatal day (PND) 20. On PND 20, pupswere weaned and housed in groups of four rats per cage. Food and tapwater were available ad libitum. All animal experiments were carriedout in accordance with the guidelines approved by the EuropeanCommunities Council Directive (86 ⁄ 609 ⁄ EEC) and by the SpanishRD 1201 ⁄ 2005.Morning doses of either 25% (v ⁄ v) ethanol (3 g ⁄ kg) in isotonic

saline, or saline, were administered intraperitoneally (i.p.) to 25-day-old pups on two consecutive days with gaps of 2 days withoutinjections, during 2 weeks. Specifically, pups were injected at PND25, 26, 29, 30, 33, 34, 37 and 38. In this way each young rat receivedeight alcohol doses simulating the binge, an intermittent drinkingpattern characteristic of young students and adolescents (Tur et al.,2003; White et al., 2006). Two groups of rats received, in addition,i.p., 30 min before the injection of the ethanol or of the saline,4 mg ⁄ kg of the anti-inflammatory compound indomethacin, suspen-ded in 0.1 mL dimethylsulphoxide and brought to a concentration of0.8 mg ⁄ mL with saline.Animals were randomly assigned to four groups (n ¼ 8–10 ⁄ group)

according to their treatments: (i) physiological saline or control; (ii)control + indomethacin; (iii) ethanol; and (iv) ethanol + indomethacin.Twenty-four hours after the last (eighth) dose of the different

treatments (PND 40), animals were killed by decapitation, brains wereremoved, and the cortex, hippocampus and cerebellum were dissectedand stored at )80 �C until use. Behavioural studies were performed intwo series of animals: adolescent (PND 41) and adult (PND 61) rats at24 h or 3 weeks after the last (eighth) ethanol administration,respectively.

Blood ethanol determination

Blood ethanol concentrations (BEC) were assessed in adolescent rats(PND 25) after a single dose of ethanol (3 g ⁄ kg, i.p.). Periodically, tailblood samples were collected in heparinized tubes and centrifuged,and ethanol was then determined using a spectrophotometric method(Sigma-Aldrich, Madrid, Spain).

Immunoblot analysis

Brain tissue from the neocortex, hippocampus and cerebellum washomogenized in RIPA buffer [phosphate-buffered saline containing1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulphate (SDS)] and protease inhibitors. Proteins were separated bySDS–polyacrylamide gel electrophoresis (PAGE) gels, transferred tonitrocellulose membranes and incubated overnight at 4 �C with thefollowing antibodies: anti-COX-2 (1 : 1000; Cayman Chem., MI,USA), anti-iNOS (1 : 250; Santa Cruz Biotechnology, Madrid, Spain),anti-GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) (1 : 5000;Chemicon, Hampshire, UK). Proteins were visualized with eitheralkaline phosphatase conjugate (Sigma-Aldrich, Madrid, Spain) orenhanced chemiluminescence system (ECL Plus, Amersham Pharma-cia Biotech., Madrid, Spain), and the intensity of the bands wasquantified with the SigmaGel image analysis software version 1.0(Jandel Scientific, Madrid, Spain).

Cell death and caspase-3 assays

Oligonucleosomal DNA fragmentation, a characteristic feature ofapoptotic cell death, was measured using a nucleosomal DNAenzyme-linked immunoabsorbent assay (Cell Death Detection ELISAkit, Roche Diagnostics, Barcelona, Spain) that quantified histone-associated DNA fragments. Brain samples from the neocortex,hippocampus and cerebellum were homogenized, centrifuged, andthe supernatant was used to quantify apoptotic cell death following themanufacturer’s instructions.Caspase-3 activity was determined using a colorimetric assay kit

(Sigma-Aldrich). Brain samples were homogenized in lysis buffer, andwere centrifuged at 10 000 g for 10 min at 4 �C. Caspase-3 wasassayed in the supernatant by adding Ac-DEVD-p-nitroanilide, andincubated for 90 min at 37 �C. Formation of the colorimetric productp-nitroanilide was measured at 405 nm, and the activity wascalculated according to the manufacturer’s instructions.

Behavioural testing

Loss of righting reflex

After a single dose of ethanol (3 g ⁄ kg, i.p.) or indomethacin(4 mg ⁄ kg, i.p.) + ethanol (3 g ⁄ kg, i.p.), each pup was placed in thesupine position in a V-shaped support until recovery. The loss ofrighting reflex (LORR), and the time between the LORR and theregain of righting reflex (RRR) was defined as the pup’s ability to rightitself on all four paws three times within a 30-s interval.

Conditional discrimination learning

The conditional discrimination learning was tested as described byMurray & Ridley (1997) in a wooden Y maze, which had three armsof an equal size (60 cm long, 11.5 cm wide and 25 cm high). The armwhere the rats were placed at the beginning of each trial wasconsidered as the start arm. The other arms, each of which had a foodcup located at the end, were considered as the choice arms. Pre-training was carried out with all rats during 4 days to familiarize themwith the maze. The whole area of the choice arms in the maze wascovered by black or white inserts (59 cm long, 11 cm wide and 25 cmhigh). In each trial, rats were rewarded for choosing the right arm, thatis, when inserts were white, and the left arm when inserts were black.The reward for the correct response consisted of four food pellets(Panlab, Barcelona, Spain) placed in the food cup at the end of thecorrect arm. If the rat made an incorrect response, it was allowed to go

542 M. Pascual et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 541–550

to the empty food cup at the end of the incorrect arm and was removedafter 5 s. Rats were trained for 10 trials per day during six sessions,with an intertrial interval of approximately 5 min in their home cage.The ratio of correct choice was determined by adding the total numberof correct choices throughout six sessions. The graphs represent thepercentages of correct choices.

Rotarod

Motor learning was performed using the rotarod test to assess theability to stay on a rotating drum. An accelerating rotarod (Jones &Roberts, 1968) was used (Ugo Basile, model 7750, Comerio, Italy). Toallow the animals to become used to the apparatus, each rat was placedin the rotarod, which was switched off for 3 min on the twoconsecutive days before testing. The rotarod speed increased from 3.5to 35 r.p.m. over 300 s. The test was conducted once a day for sixconsecutive days. The time in seconds at which each animal fell offthe rungs was recorded with a maximum cut-off of 600 s.

Object recognition test

The object recognition test was performed as described (Ennaceur &Delacour, 1988; Prickaerts et al., 2002). The apparatus consisted ofwhite cast plastic (70 · 45 · 30 cm) with sawdust on the floor, andwas located in a testing room that was dimly lit by constantillumination. The objects chosen are triple copies of square yellowplastic blocks and a round pink block, heavy enough to preventdisplacement. On the day before the test, rats were allowed to explorethe box (with no objects) for 2 min. On the day of test, a trainingsession (T1) was followed by a test session (T2) after a 1-min interval.Each session lasted 3 min. The rats were placed in the middle of thebox for T1, and were faced away from the two identical square objectsarranged in the centre of the box. The rats were removed from the boxand returned to their home cages. The objects were changed to oneclean square object (familiar object) and one round novel object (non-familiar object). One minute after T1, the rats were reintroduced intothe box for T2. Object exploration was defined as the orientation of theanimal’s snout towards the object, within a range of 2 cm or less fromthe object. Running around the object or sitting on it was not recordedas exploration. Objects were washed with ethanol after each individualtrail to equate olfactory cues.

The basic measures in the object recognition test were the timesspent by the rats to explore an object during T1 and T2 (Prickaertset al., 2002). e1 and e2 are measures of the total exploration time ofboth objects during T1 and T2, respectively. d1 was considered as anindex measure of discrimination between both the new and familiarobjects. Thus, d1 is the difference in exploration of the two objectsin T2.

Beam walking

The beam walking test was performed to assess the animal’s ability tobalance across a 1-m-long wooden beam that was raised 1.20 m abovethe ground (Metz et al., 2000; Buddeberg et al., 2004). Three types ofbeams were used as narrow pathways: a square 2.0-cm-wide beam, asquare 1.2-cm-wide beam and a round beam with a diameter of2.0 cm. Thus, the difficulty to balance across increased from beam tobeam. After training, the animals had to cross each beam three timesand were then assessed by a scoring system, as previously described(Metz et al., 2000). A score of 0 was applied for a complete inabilityto walk on the beam (the animals fell down immediately), 0.5 wasscored if the animal was able to get half way across the beam, 1 pointwas given for traversing the whole length, 1.5 points were scoredwhen stepping with the hind limbs was partially possible, and 2 points

were awarded for normal weight support and accurate foot placement.Average scores of all three beams were then added up so that amaximum of 6 points could be reached.

Statistical analysis

Results are reported as mean ± SEM. Statistical significance forWestern blots and cell death was determined by a one-way anova

test. Data of conditional discrimination learning, object recognitionand narrow beam tasks were analysed with separate one-way anova

and a between variable ‘Treatment’ with four levels (control,control + indomethacin, ethanol, and ethanol + indomethacin) forboth animal series: adolescent (PND 39) or adult (PND 60). Therotarod test data were analysed with a two-way anova and a betweenvariable ‘Treatment’ with four levels (control, control + indomethacin,ethanol, and ethanol + indomethacin) and a variable within ‘Days’with two levels (first and sixth) for both animal series: adolescent(PND 39) or adult (PND 60). Post hoc comparisons were performedwith the Newman–Keuls test.

Results

BECs and hypnotic sensitivity to alcohol

After a single dose of ethanol, BEC resulted in a peak of210 ± 11 mg ⁄ dL at 30 min, post-injection. This was followed by agradual decline to a concentration close to zero at the 540-minsampling point. Indomethacin administration neither affected BEC(peak 205 ± 9 mg ⁄ dL at 30 min) nor the kinetics of ethanolelimination.Hypnotic sensitivity to alcohol was measured by determining the

duration of the LORR. Ethanol administration resulted in a rapid onsetof the LORR (25-day-old rats, ethanol group: 96 ± 14 s; indometh-acin + ethanol group: 99 ± 13 s; n.s.). The duration of the LORR orRRR was not significantly affected by indomethacin administration(ethanol group: 58 ± 10 min; indomethacin + ethanol: 62 ± 8 min;n.s.).

Intermittent ethanol binge treatment enhances inflammatorymediators in the brain of adolescent rats: effect of indomethacinadministration

Our recent results demonstrated that chronic ethanol treatment inducesbrain inflammatory damage by increasing the production of cytokinesand inflammatory mediators such as COX-2 and iNOS, and cell death(Valles et al., 2004). Therefore, in order to assess whether intermittentethanol binge treatment can also induce inflammatory mediators in theadolescent rat’s brain, we measured the levels of COX-2 and iNOS indifferent brain areas from animals intermittently treated with ethanolover a 15-day period. The levels of these proteins were assessed byWestern blot at the end of the intermittent ethanol treatment, andspecifically 24 h after the last ethanol administration. As Figs 1 and 2illustrate, ethanol binge treatment significantly increased the levels ofCOX-2 and iNOS in the neocortex, hippocampus and cerebellum,when compared with the control group.Furthermore, when indomethacin was administered 30 min prior

to ethanol treatment, this anti-inflammatory compound was noted tocompletely suppress the upregulation of COX-2 and iNOS inducedby ethanol in both the neocortex and hippocampus, although only apartial suppression in the levels of COX-2, or no effects in iNOS,were noted in the cerebellum (Figs 1 and 2). There was no

Ethanol induces inflammation and behaviour dysfunctions 543


significant modification noted in the expression of these proteins incomparison with the controls when indomethacin was administeredalone.

Fig. 1. Western blotting and densitometric analysis of cyclooxygenases-2(COX-2) in the neocortex (A), hippocampus (B) and cerebellum (C), obtained24 h after the last ethanol administration from the four animal groups: controlor physiological saline (Ctrol); 4 mg ⁄ kg indomethacin (Ctrol + Indom);3 g ⁄ kg ethanol (EtOH); ethanol (3 g ⁄ kg) and indomethacin (4 mg ⁄ kg)(EtOH + Indom). Blots were redeveloped with anti-GAPDH to show equalprotein loading. A representative immunoblot is shown. Data are themean ± SEM of four independent experiments. (A and B) **P < 0.01 vs. theother groups; (C) *P < 0.05 vs. the other groups, ##P < 0.01 vs. control.

Fig. 2. Western blotting and densitometric analysis of inducible nitric oxidesynthase (iNOS) in the neocortex (A), hippocampus (B) and cerebellum (C),obtained 24 h after the last ethanol administration from the four animalgroups: control or physiological saline (Ctrol); 4 mg ⁄ kg indomethacin(Ctrol + Indom); 3 g ⁄ kg ethanol (EtOH); ethanol (3 g ⁄ kg) and indomethacin(4 mg ⁄ kg) (EtOH + Indom). Blots were redeveloped with anti-GAPDH toshow equal protein loading. A representative immunoblot is shown. Data arethe mean ± SEM of four independent experiments. (A) *P < 0.05 vs. theother groups; (B) **P < 0.01 vs. the other groups; (C) *P < 0.05 vs. controlgroup.



Intermittent ethanol treatment induces cell death in the brain ofadolescent rats: effect of indomethacinWe next evaluated whether intermittent ethanol treatment can causeneural damage and increased cell death. The levels of caspase-3activity and apoptotic cell death, assessed by the determination ofhistone-associated DNA fragments, were determined in the neocortex,hippocampus and cerebellum from the brain of rats 24 h after the lastdose (eighth dose) of ethanol treatment. Figure 3 illustrates thatintermittent ethanol treatment significantly enhances both apoptosisand caspase-3 activity in the neocortex, hippocampus and cerebellum

when compared with the control group (P < 0.05). We have to pointout that a single dose of ethanol (3 g ⁄ kg) neither significantly changedthe levels of the COX-2 and iNOS expressions nor apoptotic cell death(data not shown), suggesting that brain damage occurs after severaldoses of ethanol.Notably, administering indomethacin before ethanol treatment

completely abolished ethanol-induced cell death in the hippocampus(P < 0.05; Fig. 3B), although this compound partially rescuesethanol-induced cell death in the neocortex and cerebellum (Fig. 3Aand C).

Fig. 3. Effects of ethanol and ⁄ or indomethacin on apoptotic cell death and caspase-3 activity in different brain regions. Tissue extracts from the neocortex (A),hippocampus (B) and cerebellum (C) obtained 24 h after the last ethanol administration from the four animal groups: control or physiological saline (Ctrol);4 mg ⁄ kg indomethacin (Ctrol + Indom); 3 g ⁄ kg ethanol (EtOH); ethanol (3 g ⁄ kg) and indomethacin (4 mg ⁄ kg) (EtOH + Indom). Data are the mean ± SEM offour different experiments. (A) *P < 0.05 vs. control; (B) *P < 0.05 vs. the other groups; (C) *P < 0.05 vs. control group.



Multiple binge alcohol treatments during adolescence causeslong-term behavioural dysfunctions: indomethacin effects

We then assessed whether ethanol-induced iNOS and COX-2expressions and cell death in the neocortex, hippocampus andcerebellum were associated with the neurobehavioural dysfunctionin adolescent animals, and whether these dysfunctions were permanentor caused long-lasting behavioural outcomes. To answer thesequestions, we performed several tests associated with the integrityof these brain areas. Specifically, conditional discrimination learning(Murray & Ridley, 1999) and object recognition task (Ennaceur &Aggleton, 1997; Eichenbaum, 2000) were assessed to evaluate thecortical and hippocampal functions, and rotarod and beam-walkingperformances were used to test motor coordination as they are tasksassociated with cerebellar functions (Yamamoto et al., 2003).

Conditional discrimination learning

Conditional discrimination learning was assessed in a wooden Ymaze. The correct choice ratio was determined by adding the totalnumber of correct choices throughout six sessions. During the trainingsessions, a significant reduction in the percentage of correct choices(F3,24 ¼ 2.88; P < 0.05) was noted in ethanol-treated adolescent ratswhen compared with adolescent control animals (Fig. 4A). Further-more, the anova of the data collected from adult rats revealed asignificant effect of the variable treatment (F3,28 ¼ 6.135;P < 0.0024), suggesting that ethanol causes long-term effects in

learning ability, which were also observed at the adult stage (Fig. 4B).Post hoc comparison further confirmed these results, and indicatedthat the ethanol-treated group achieved less correct responses than theother groups (P < 0.01).Our results also demonstrated that indomethacin treatment abol-

ished the deficits in the learning ability induced by ethanol in bothadolescent and adult animals.

Rotarod

Motor learning was performed using the rotarod test to assess theability to stay on a rotating drum. The anova carried out with data ofadolescent rats showed that although the variable treatment was notsignificant (F3,30 ¼ 1.061; n.s.) during the first trial, all groupsshowed significant improvement across days (F1,30 ¼ 287.092;P < 0.0001). On the sixth day, however, the ethanol-treated groupspent less time in the rotarod than the other groups (F3,30 ¼ 5.887;P < 0.0028). The post hoc comparisons analysis further confirmedthese results (P < 0.01). Indomethacin treatment improved the effectsof ethanol in motor learning in the last trial (Fig. 5A).The anova performed with data of adult rats (Fig. 5B) showed that

only the variable Days was significant (F1,35 ¼ 86.390; P < 0.0001),indicating that the performance of all groups improved with training.

Fig. 4. Effects of ethanol and ⁄ or indomethacin on the conditional discrim-ination learning task. Bars represent the mean (± SEM, n ¼ 8–10) percentageof the correct choice in the four animal groups following the treatment receivedover 2 weeks (two consecutive days of injections with gaps of 2 days withoutinjections): control or physiological saline (Ctrol); 4 mg ⁄ kg indomethacin(Ctrol + Indom); 3 g ⁄ kg ethanol (EtOH); ethanol (3 g ⁄ kg) and indomethacin(4 mg ⁄ kg) (EtOH + Indom). (A) Rats were tested at the adolescent stage(PND 39) 24 h after the last ethanol administration. (B) Rats were tested at theadult stage (PND 60) 3 weeks after the last treatment administration.*P < 0.05, **P < 0.01 significant difference in relation to control group.

Fig. 5. Effects of ethanol and ⁄ or indomethacin on the rotarod task. Barsrepresent the mean (± SEM, n ¼ 8–10) time in seconds spent in the rotarodduring the first or the last (sixth) trial in four animal groups, following treatmentreceived over 2 weeks (two consecutive days of injections with gaps of 2 dayswithout injections): control or physiological saline (Ctrol); 4 mg ⁄ kg indo-methacin (Ctrol + Indom); 3 g ⁄ kg ethanol (EtOH); ethanol (3 g ⁄ kg) andindomethacin (4 mg ⁄ kg) (EtOH + Indom). (A) Rats were tested at theadolescent stage (PND 39) 24 h after the last treatment administration.(B) Rats were tested at the adult stage (PND 60) 3 weeks after the lasttreatment administration. **P < 0.01 significant difference in relation to controlgroup.



However, ethanol treatment (F3,35 ¼ 0.412; n.s.) and the interactionTreatment · Days (F3,35 ¼ 1.413; n.s.) were not significant.

Object recognition test

The results of the object recognition test of treatments with ethanoland indomethacin in adolescent rats are summarized in Table 1. Theanova performed with data obtained in the d1 measure in adolescentrats showed a significant reduction in the ethanol-treated group(F3,30 ¼ 3.289; P < 0.0341). Post hoc comparison indicates that theethanol-treated group differs from the groups treated with saline orindomethacin or indomethacin + ethanol (P < 0.05), suggesting thatethanol-treated animals did not discriminate between novel andfamiliar objects. No differences were found between the varioustreatments in the total exploration time in T1 (e1) (F3,30 ¼ 1.932;n.s.), nor were there differences between the treatments in the totalexploration time in T2 (e2) (F3,30 ¼ 1.747; n.s.).

The anova of the data obtained in the same measure in adult rats(Table 2) also revealed that the ethanol-treated group showed asignificant reduction in the discrimination index, d1 (F3,30 ¼ 7.269;P < 0.0008). These data were further confirmed by the post hocanalysis, which indicated that the ethanol-treated group differs fromthe other groups (P < 0.01). The anova carried out with dataobtained in e1 (F3,30 ¼ 2.05; P < 0.128) and e2 (F3,30 ¼ 2.302; n.s.)measures in adult rats were not significant.

The results also demonstrated that indomethacin protects against thereduction in the discrimination index induced by intermittent ethanoltreatment during the adolescence stage.

Beam walking

We finally assessed the effect of ethanol on the ability to adapt to thechallenging motor task by using narrow beam walking. After training,

animals had to cross a different type of beam three times and were thenassessed by a scoring system. The anova of the data demonstratedthat ethanol treatment significantly reduces the score in bothadolescent (F3,28 ¼ 37.142; P < 0.0001) and adult (F3,30 ¼ 12.857;P < 0.0001) animals (Fig. 6A and B). Post hoc analysis indicates thatthe group treated with ethanol + indomethacin presented a higherscore than the group treated with ethanol (P < 0.01), although theethanol + indomethacin group showed a lower score than the groupstreated with saline or indomethacin (P < 0.01), suggesting that thiscompound partially restores the effects of ethanol on beam walking inadolescent rats.

Discussion

Cumulative evidence revealed the special susceptibility of theadolescent brain to both ethanol-induced neurotoxicity and cognitiveimpairments. The mechanisms underlying these harmful effects ofethanol are presently unknown. In the present study we demonstratethat intermittent ethanol administration during the adolescence stageincreases the levels of COX-2 and iNOS, enhances neural cell death inseveral brain regions, and causes long-lasting neurobehaviouralimpairments. Furthermore, administration of the COX-2 inhibitor,indomethacin, not only abolishes the enhancement in apoptosis andthe upregulation of both COX-2 and iNOS expressions, but alsoameliorates the ethanol-induced neurobehavioural deficits.The roles of COX-2 and iNOS in the inflammatory processes and in

oxidative stress are well established, and elevated levels of theseenzymes occur during brain damage, such as in ischaemia, excitotox-icity and neurodegenerative diseases (Heales et al., 1999; O’Banion,1999; Yamada et al., 1999; Campbell, 2004). In most of these disorders,elevated levels of COX-2 and iNOS are associated with an increase inneural death and also neurobehavioural dysfunctions. For example,COX-2 transgenic mice that overexpress this protein displayed signi-ficant neurobehavioural changes with a parallel age-dependent increase

Table 1. Results of treatment in adolescent rats (PND 39) on the measures of the object recognition test

ControlControl +indomethacin Ethanol

Ethanol +indomethacin

Exploration time (s) during: the first (e1) and second trial (e2)e1 23.75 ± 4.47 25.50 ± 4.23 15.11 ± 1.44 20.56 ± 2.75e2 19.25 ± 6.08 18.00 ± 2.41 7.89 ± 2.00 16.11 ± 4.25

Index of discrimination (d1) between new and familiar objectsd1 8.00 ± 2.51 9.00 ± 2.75 1.22 ± 0.79* 5.67 ± 1.47

Data represent the mean ± SEM (n ¼ 8–10) in adolescent rats (PND 39) tested 24 h after the last administration of the following treatments: control or physiologicalsaline; 4 mg ⁄ kg indomethacin; 3 g ⁄ kg ethanol; ethanol (3 g ⁄ kg) + indomethacin (4 mg ⁄ kg). *P < 0.05 significant difference with respect to control group.

Table 2. Results of treatment in adult rats (PND 60) on the measures of the objects recognition test

ControlControl +indomethacin Ethanol

Ethanol +indomethacin

Total exploration time (s) during the first (e1) and second trial (e2)e1 22.25 ± 3.35 23.25 ± 5.93 12.44 ± 2.27 15.89 ± 2.44e2 18.06 ± 3.04 22.38 ± 6.30 9.28 ± 2.49 14.39 ± 1.96

Index of discrimination (d1) between new and familiar objectsd1 10.19 ± 1.43 8.38 ± 1.10 2.06 ± 1.89** 8.72 ± 1.76

Data represent the mean (± SEM, n ¼ 8–10) in adult rats (PND 60) tested 3 weeks after the last administration of the following treatments: control or physiologicalsaline; 4 mg ⁄ kg indomethacin; 3 g ⁄ kg ethanol; ethanol (3 g ⁄ kg) + indomethacin (4 mg ⁄ kg). **P < 0.01 significant difference with respect to control group.



in neuronal apoptosis (Andreasson et al., 2001). Similarly, intermittenthypoxia causes spatial learning deficits (Li et al., 2004), and a correlationhas been observed between iNOS induction and an increase in neuralapoptosis (Goldbart et al., 2003). Although the initiating eventsresponsible for these pathological changes remain unclear, increasingevidence demonstrates that brain injury is associated with both glialactivation and cytokine production, which strongly induces iNOS andCOX-2 expressions leading to massive uncontrolled toxic compoundsthat would damage neighbouring neurons (Brown & Borutaite, 1999;Andreasson & Kaufmann, 2002).Our recent results demonstrate that ethanol is able to induce a rapid

response in glial cells by upregulating the COX-2 and iNOSexpressions and the NO production, and that these effects aremediated via NFkB (Blanco et al., 2004, 2005). Accordingly, chronicethanol treatment stimulates glial cells, triggering signalling pathwaysin the brain that lead to the production and expression of cytokines andinflammatory mediators, and also to cell death (Valles et al., 2004).The present study provides further evidence that some of theneurotoxic effects of ethanol during the juvenile ⁄ adolescence stageare mediated by inflammatory processes. Thus, our results demon-strate that intermittent ethanol intake during adolescence can inducecell death, and that these effects are associated with elevated levels ofiNOS and COX-2 in the neocortex, hippocampus and cerebellum.Supporting the role of inflammation in the ethanol-induced braindamage, administration of indomethacin abolished the ethanol-induced increase in cell death and the upregulation of iNOS and

COX-2. Consistent with these findings, it has been demonstrated thatindomethacin exerts protective effects after glial cell activation(Shibata et al., 2003) and cerebral ischaemia (Kluska et al., 2005).Although the above mechanism of ethanol-induced neurotoxicity by

repeated periods of ethanol intoxication could also occur in adults,recent evidence indicates that adolescent animals are more sensitivethan adults to the disruptive effects of acute ethanol exposure in bothcognitive processes and ethanol-induced brain injury (Crews et al.,2000; White & Swartzwelder, 2005). For example, ethanol-induceddisruptions in long-term potentiation are more robust in hippocampalslices from periadolescent rats than in slices from the adulthippocampus (Swartzwelder et al., 1995; Pyapali et al., 1999).Adolescent animals also show greater impairments in spatial memorythan adult rats (Markwiese et al., 1998; Sircar & Sircar, 2005) and,unlike adult rats, performance deficits persist for some weeks afterethanol treatment (Sircar & Sircar, 2005). Conversely, juvenileanimals are less sensitive to the sedation and temperature regulationeffects of ethanol than adults (Ristuccia & Spear, 2004). Binge-likedrinking among adolescent rats also increases the anxiety-relatedbehaviour (Popovic et al., 2004).The high sensitivity of the juvenile ⁄ adolescent brain to some

harmful effects of ethanol might be due to the fact that adolescence is astage of brain development at which the brain undergoes significantchanges in its organization and functions, which underlies thematuration of cognitive processing (Hiller-Sturmhofel & Swartz-welder, 2005; Toga et al., 2006). Considering that the developingbrain is highly vulnerable to the damaging effects of ethanol, and thatthese effects are usually irreversible (for a review, see Guerri, 2002), itmight be expected that binge drinking during adolescence could affectbrain maturation by altering the developmental plasticity, leading tolifelong impairments (for a review, see Hiller-Sturmhofel &Swartzwelder, 2005). Indeed, the present results show that intermittentbinge-like drinking during adolescence can cause significant short-and long-term neurobehavioural alterations that are associated withboth the induction of inflammatory mediators and apoptosis in theneocortex, hippocampus and cerebellum. We show that ethanolexposure during the juvenile ⁄ adolescent stage impairs cognitiveprocesses, as reflected by deficits in both conditional discriminationand object recognition tasks. Defects in these tasks were observed inboth young and adult rats exposed to ethanol during the periadolescentperiod, indicating long-lasting changes in the functional brain activity.Because conditional learning is sensitive to lesions in the hippocam-pus (Murray & Ridley, 1999) and deficits in the object discriminativememory have been associated with hippocampal and corticalimpairments (Ennaceur & Aggleton, 1997; Eichenbaum, 2000), theseresults suggest that defects in the performance of these tasks may beassociated with ethanol-induced hippocampal and cortical damage.Indeed, several studies reported the vulnerability of the hippocampusand its functions to the effects of ethanol during adolescence (DeBellis et al., 2000; White & Swartzwelder, 2005). In addition, Crewset al. (2000) reported ethanol-induced damage in the frontal corticalareas of juvenile ⁄ adolescent rats, but not in adult rats. Frontal corticalregions are known to undergo developmental reorganization andfunctions, including an intense rewiring that underlies maturation andcognitive processing (Giedd, 2004; Toga et al., 2006).Cognitive deficits compatible with prefrontal cortex and hippocampal

dysfunctions have been observed in human adolescentswith alcohol-usedisorders (Brown et al., 2000; De Bellis et al., 2000, 2005; Brown &Tapert, 2004). These observations demonstrated that heavy drinkingduring adolescence is associated with poorer performance on testsrequiring attention skills, andwith deficits in retrieval of verbal and non-verbal information as well as in visuospatial functioning (Brown et al.,

Fig. 6. Effects of ethanol and ⁄ or indomethacin on the beam walking task.Bars represent the mean (± SEM, n ¼ 8–10) score obtained in the narrow beamtest conducted by the four animal groups, following treatment received over2 weeks (two consecutive days of injections with gaps of 2 days withoutinjections): control or physiological saline (Ctrol); 4 mg ⁄ kg indomethacin(Ctrol + Indom); 3 g ⁄ kg ethanol (EtOH); ethanol (3 g ⁄ kg) and indomethacin(4 mg ⁄ kg) (EtOH + Indom). (A) Rats were tested at the adolescent stage(PND 39) 24 h after the last treatment administration. (B) Rats were tested atthe adult stage (PND 60) 3 weeks after the last treatment administration.**P < 0.01, significant difference in relation to the control group, ##P < 0.01significant difference in relation to the control group.



2000; Brown & Tapert, 2004). Alcohol withdrawal over the teen yearsappears to contribute to deterioration in functioning in visual tasks(Brown et al., 2000; Tapert et al., 2002). Some of these dysfunctions,such as the visual learning deficits and neurocognitive functioning, areevident even after 4 years of abstention (Tapert et al., 1999;Brown et al.,2000; Brown & Tapert, 2004). Notably, visuospatial memory abilitiesare associated with the frontal lobe system (Sowell et al., 2001), anddysfunctions in the prefrontal cortex occur in alcoholics and have beenassociated with repeated experience of alcohol withdrawal, impairedcognitive function and compulsive behaviour, which can predispose toalcohol abuse (Lyvers, 2000; Moselhy et al., 2001; Duka et al., 2003).Therefore, ethanol-induced prefrontal cortex impairments (Crews et al.,2000) in adolescents might underlie the cognitive dysfunctions andmight also predispose to alcohol abuse and dependence (Grant, 1998).Indeed, recent studies suggest that a smaller prefrontal cortex isassociated with early-onset drinking in adolescents with co-morbidmental disorders (De Bellis et al., 2005).

Our results also show that intermittent ethanol intake during thejuvenile ⁄ adolescence stage affects the motor function, as demonstra-ted by impaired performance on rotarod and beam walking, testsusually associated with cerebellar functions (Yamamoto et al., 2003),although cortical disruptions also impair beam-walking performance(Hofferer & Cassel, 1996; Allen et al., 2000). However, although wefound impairment on the rotarod performance in ethanol-exposedadolescent rats, this effect did not persist in adulthood, suggesting thatsome plasticity can occur at this stage to compensate the effects ofethanol (Toga et al., 2006).

Finally, indomethacin administration not only blocks neural celldeath, but also attenuates short- and long-term behavioural deficitsinduced by intermittent ethanol exposure during adolescence, which isin line with the proposed role of inflammation in ethanol-inducedbrain damage and behavioural effects.

In summary, we show that intermittent ethanol treatment duringadolescence triggers the induction of iNOS and COX-2, and also celldeath in the neocortex, hippocampus and cerebellum by impairing thenormal brain maturation and plasticity to cause long-lasting neuro-behavioural deficits. Our results suggest that inflammation plays aprominent role in the pathophysiological mechanisms that mediateneurobehavioural deficits.

Acknowledgements

We would like to thank M. March, M.C. Garcıa-Minguillan, M.A. Perez-Arago and M.J. Agullo for their excellent technical assistance. This work wassupported by grants from the Spanish Ministry of Education and Science(SAF 2006-02178 and SEJ2005-00316), the Spanish Ministry of Heath (FIS),the RTA Network (G03 ⁄ 005), Direccion General de Drogodependencias(GV), FEPAD (GV) and the Fundacion de Investigacion Medica MutuaMadrilena.

Abbreviations

BEC, blood ethanol concentration; COX-2, cyclooxygenase-2; GAPDH,glyceralaldehyde-3-phosphate-dehydrogenase; iNOS, inducible nitric oxidesynthase; LORR, loss of righting reflex; NSAID, non-steroidal anti-inflamma-tory drugs; PND, postnatal day; RRR, regain of righting reflex; SDS, sodiumdodecyl sulphate.

References

Allen, G.V., Gerami, D. & Esser, M.J. (2000) Conditioning effects of repetitivemild neurotrauma on motor function in an animal model of focal brain injury.Neuroscience, 99, 93–105.

Andreasson, K.I. & Kaufmann, W.E. (2002) Role of immediate early geneexpression in cortical morphogenesis and plasticity. Results Probl. CellDiffer., 39, 113–137.

Andreasson, K.I., Savonenko, A., Vidensky, S., Goellner, J.J., Zhang, Y.,Shaffer, A., Kaufmann, W.E., Worley, P.F., Isakson, P. & Markowska, A.L.(2001) Age-dependent cognitive deficits and neuronal apoptosis in cyclo-oxygenase-2 transgenic mice. J. Neurosci., 21, 8198–8209.

Blanco, A.M., Pascual, M., Valles, S.L. & Guerri, C. (2004) Ethanol-inducediNOS and COX-2 expression in cultured astrocytes via NF-kappa B.Neuroreport, 15, 681–685.

Blanco, A.M., Valles, S.L., Pascual, M. & Guerri, C. (2005) Involvement ofTLR4 ⁄ type I IL-1 receptor signaling in the induction of inflammatorymediators and cell death induced by ethanol in cultured astrocytes.J. Immunol., 175, 6893–6899.

Brown, G.C. & Borutaite, V. (1999) Nitric oxide, cytochrome c andmitochondria. Biochem. Soc. Symp., 66, 17–25.

Brown, S.A. & Tapert, S.F. (2004) Adolescence and the trajectory of alcoholuse: basic to clinical studies. Ann. N.Y. Acad. Sci., 1021, 234–244.

Brown, S.A., Tapert, S.F., Granholm, E. & Delis, D.C. (2000) Neurocognitivefunctioning of adolescents: effects of protracted alcohol use. Alcohol. Clin.Exp. Res., 24, 164–171.

Buddeberg, B.S., Kerschensteiner, M., Merkler, D., Stadelmann, C. &Schwab, M.E. (2004) Behavioral testing strategies in a localized animalmodel of multiple sclerosis. J. Neuroimmunol., 153, 158–170.

Campbell, A. (2004) Inflammation, neurodegenerative diseases, and environ-mental exposures. Ann. N.Y. Acad. Sci., 1035, 117–132.

Crews, F.T., Braun, C.J., Hoplight, B., Switzer, R.C. 3rd & Knapp, D.J. (2000)Binge ethanol consumption causes differential brain damage in youngadolescent rats compared with adult rats. Alcohol. Clin. Exp. Res., 24, 1712–1723.

Crews, F.T., Collins, M.A., Dlugos, C., Littleton, J., Wilkins, L., Neafsey, E.J.,Pentney, R., Snell, L.D., Tabakoff, B., Zou, J. & Noronha, A. (2004)Alcohol-induced neurodegeneration: when, where and why? Alcohol. Clin.Exp. Res., 28, 350–364.

Dahl, R.E. (2004) Adolescent brain development: a period of vulnerabilitiesand opportunities. Keynote address. Ann. N.Y. Acad. Sci., 1021, 1–22.

De Bellis, M.D., Clark, D.B., Beers, S.R., Soloff, P.H., Boring, A.M., Hall, J.,Kersh, A. & Keshavan, M.S. (2000) Hippocampal volume in adolescent-onset alcohol use disorders. Am. J. Psychiatry, 157, 737–744.

De Bellis, M.D., Narasimhan, A., Thatcher, D.L., Keshavan, M.S., Soloff, P.& Clark, D.B. (2005) Prefrontal cortex, thalamus, and cerebellar volumesin adolescents and young adults with adolescent-onset alcohol usedisorders and comorbid mental disorders. Alcohol. Clin. Exp. Res., 29,1590–1600.

Duka, T., Townshend, J.M., Collier, K. & Stephens, D.N. (2003) Impairment incognitive functions after multiple detoxifications in alcoholic inpatients.Alcohol. Clin. Exp. Res., 27, 1563–1572.

Eichenbaum, H. (2000) A cortical-hippocampal system for declarative memory.Nat. Rev., 1, 41–50.

Ennaceur, A. & Aggleton, J.P. (1997) The effects of neurotoxic lesions of theperirhinal cortex combined to fornix transection on object recognitionmemory in the rat. Behav. Brain Res., 88, 181–193.

Ennaceur, A. & Delacour, J. (1988) A new one-trial test for neurobiologicalstudies of memory in rats. 1: behavioral data. Behav. Brain Res., 31, 47–59.

Giedd, J.N. (2004) Structural magnetic resonance imaging of the adolescentbrain. Ann. N.Y. Acad. Sci., 1021, 77–85.

Goldbart, A., Row, B.W., Kheirandish, L., Schurr, A., Gozal, E., Guo, S.Z.,Payne, R.S., Cheng, Z., Brittian, K.R. & Gozal, D. (2003) Intermittenthypoxic exposure during light phase induces changes in cAMP responseelement binding protein activity in the rat CA1 hippocampal region: watermaze performance correlates. Neuroscience, 122, 585–590.

Grant, B.F. (1998) The impact of a family history of alcoholism on therelationship between age at onset of alcohol use and DSM-IV alcoholdependence: results from the National Longitudinal Alcohol EpidemiologicSurvey. Alcohol. Health Res. World, 22, 144–147.

Guerri, C. (2002) Mechanisms involved in central nervous system dysfunctionsinduced by prenatal ethanol exposure. Neurotox. Res., 4, 327–335.

Haberly, L.B. & Bower, J.M. (1989) Olfactory cortex: model circuit for studyof associative memory? Trends Neurosci., 12, 258–264.

Harper, C. & Matsumoto, I. (2005) Ethanol and brain damage. Curr. Opin.Pharmacol., 5, 73–78.

Heales, S.J., Bolanos, J.P., Stewart, V.C., Brookes, P.S., Land, J.M. &Clark, J.B. (1999) Nitric oxide, mitochondria and neurological disease.Biochim. Biophys. Acta, 1410, 215–218.



Hiller-Sturmhofel, S. & Swartzwelder, H.S. (2005) Alcohol’s effects on theadolescent brain: what can be learned from animal models. Alcohol. Res.Health, 28, 213–221.

Hofferer, E. & Cassel, J.C. (1996) A comparison of the effects of two fimbria-fornix lesion techniques on beam-walking performance in the rat: aspirationversus electrolysis. Behav. Brain Res., 74, 175–180.

Iadecola, C., Niwa, K., Nogawa, S., Zhao, X., Nagayama, M., Araki, E.,Morham, S. & Ross, M.E. (2001) Reduced susceptibility to ischemic braininjury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc. Natl Acad. Sci. USA, 98, 1294–1299.

Jones, B.J. & Roberts, D.J. (1968) The quantitative measurement of motorincoordination in naive mice using an accelerating rotarod. J. Pharm.Pharmacol., 20, 302–304.

Kluska, M.M., Witte, O.W., Bolz, J. & Redecker, C. (2005) Neurogenesis in theadult dentate gyrus after cortical infarcts: effects of infarct location,N-methyl-D-aspartate receptor blockade and anti-inflammatory treatment.Neuroscience, 135, 723–735.

Knott, C., Stern, G. & Wilkin, G.P. (2000) Inflammatory regulators inParkinson’s disease: iNOS, lipocortin-1, and cyclooxygenases-1 and -2.Mol. Cell Neurosci., 16, 724–739.

Li, R.C., Row, B.W., Kheirandish, L., Brittian, K.R., Gozal, E., Guo, S.Z.,Sachleben, L.R. & Gozal, D. (2004) Nitric oxide synthase and intermittenthypoxia-induced spatial learning deficits in the rats. Neurobiol. Dis., 17, 44–53.

Lyvers, M. (2000) ‘Loss of control’ in alcoholism and drug addiction: aneuroscientific interpretation. Exp. Clin. Psychopharmacol., 8, 225–249.

Markwiese, B.J., Acheson, S.K., Levin, E.D., Wilson, W.A. & Swartzwelder,H.S. (1998) Differential effects of ethanol on memory in adolescent and adultrats. Alcohol. Clin. Exp. Res., 22, 416–421.

Metz, G.A., Merkler, D., Dietz, V., Schwab, M.E. & Fouad, K. (2000) Efficienttesting of motor function in spinal cord injured rats. Brain Res., 883,165–177.

Minghetti, L. (2004) Cyclooxygenase-2 (COX-2) in inflammatory anddegenerative brain diseases. J. Neuropathol. Exp. Neurol., 63, 901–910.

Monti, P.M., Miranda, R. Jr, Nixon, K., Sher, K.J., Swartzwelder, H.S., Tapert,S.F., White, A. & Crews, F.T. (2005) Adolescence: booze, brains, andbehavior. Alcohol. Clin. Exp. Res., 29, 207–220.

Moselhy, H.F., Georgiou, G. & Kahn, A. (2001) Frontal lobe changes inalcoholism: a review of the literature. Alcohol Alcohol., 36, 357–368.

Murray, T.K. & Ridley, R.M. (1997) The effect of dizocilpine (MK-801) onconditional discrimination learning in the rat. Behav. Pharmacol., 8,383–388.

Murray, T.K. & Ridley, R.M. (1999) The effect of excitotoxic hippocampallesions on simple and conditional discrimination learning in the rat. Behav.Brain Res., 99, 103–113.

O’Banion, M.K. (1999) Cyclooxygenase-2: molecular biology, pharmacology,and neurobiology. Crit. Rev. Neurobiol., 13, 45–82.

Popovic, M., Caballero-Bleda, M., Puelles, L. & Guerri, C. (2004) Multiplebinge alcohol consumption during rat adolescence increases anxiety butdoes not impair retention in the passive avoidance task. Neurosci. Lett., 357,79–82.

Prendergast, M.A., Harris, B.R., Mullholland, P.J., Blanchard, J.A. 2nd,Gibson, D.A., Holley, R.C. & Littleton, J.M. (2004) Hippocampal CA1region neurodegeneration produced by ethanol withdrawal requires activa-tion of intrinsic polysynaptic hippocampal pathways and function of N-methyl-D-aspartate receptors. Neuroscience, 124, 869–877.

Prickaerts, J., Van Staveren, W.C., Sik, A., Markerink-van Ittersum, M.,Niewohner, U., van der Staay, F.J., Blokland, A. & de Vente, J. (2002)Effects of two selective phosphodiesterase type 5 inhibitors, sildenafil andvardenafil, on object recognition memory and hippocampal cyclic GMPlevels in the rat. Neuroscience, 113, 251–361.

Pyapali, G.K., Turner, D.A., Wilson, W.A. & Swartzwelder, H.S. (1999) Ageand dose-dependent effects of ethanol on the induction of hippocampal long-term potentiation. Alcohol., 19, 107–111.

Ristuccia, R.C. & Spear, L.P. (2004) Adolescent ethanol sensitivity:hypothermia and acute tolerance. Ann. N.Y. Acad. Sci., 1021, 445–447.

Shibata, H., Katsuki, H., Nishiwaki, M., Kume, T., Kaneko, S. & Akaike, A.(2003) Lipopolysaccharide-induced dopaminergic cell death in rat midbrainslice cultures: role of inducible nitric oxide synthase and protection byindomethacin. J. Neurochem., 86, 1201–1212.

Sircar, R. & Sircar, D. (2005) Adolescent rats exposed to repeated ethanoltreatment show lingering behavioral impairments. Alcohol. Clin. Exp. Res.,29, 1402–1410.

Sowell, E.R., Delis, D., Stiles, J. & Jernigan, T.L. (2001) Improved memoryfunctioning and frontal lobe maturation between childhood and adolescence:a structural MRI study. J. Int. Neuropsychol. Soc., 7, 312–322.

Swartzwelder, H.S., Wilson, W.A. & Tayyeb, M.I. (1995) Age-dependentinhibition of long-term potentiation by ethanol in immature versus maturehippocampus. Alcohol. Clin. Exp. Res., 19, 1480–1485.

Tapert, S.F., Granholm, E., Leedy, N.G. & Brown, S.A. (1999) Neuropsycho-logical correlates of adolescent substance abuse: four-year outcomes. J. Int.Neuropsychol. Soc., 5, 481–493.

Tapert, S.F., Granholm, E., Leedy, N.G. & Brown, S.A. (2002) Substance useand withdrawal: neuropsychological functioning over 8 years in youth. J. Int.Neuropsychol. Soc., 8, 873–883.

Toga, A.W., Thompson, P.M. & Sowell, E.R. (2006) Mapping brain maturation.Trends Neurosci., 29, 148–159.

Tur, J.A., Puig, M.S., Pons, A. & Benito, E. (2003) Alcohol consumptionamong school adolescents in Palma de Mallorca. Alcohol Alcohol., 38, 243–248.

Valles, S.L., Blanco, A.M., Pascual, M. & Guerri, C. (2004) Chronic ethanoltreatment enhances inflammatory mediators and cell death in the brain and inastrocytes. Brain Pathol., 14, 365–371.

White, A.M., Kraus, C.L. & Swartzwelder, H. (2006) Many college freshmendrink at levels far beyond the binge threshold. Alcohol. Clin. Exp. Res., 30,1006–1010.

White, A.M. & Swartzwelder, H.S. (2005) Age-related effects of alcohol onmemory and memory-related brain function in adolescents and adults. RecentDev. Alcohol., 17, 161–176.

Willard, L.B., Hauss-Wegrzyniak, B., Danysz, W. & Wenk, G.L. (2000)Inhibition by free radical scavengers and by cyclooxygenase inhibitors ofpial arteriolar abnormalities from concussive brain injury in cats. Circ. Res.,48, 95–103.

Yamada, K., Komori, Y., Tanaka, T., Senzaki, K., Nikai, T., Sugihara, H.,Kameyama, T. & Nabeshima, T. (1999) Brain dysfunction associated withand induction of nitric oxide synthase following an intracerebral injection oflipopolysaccharide in rats. Neuroscience, 88, 281–294.

Yamamoto, M., Wada, N., Kitabatake, Y., Watanabe, D., Anzai, M., Yokoyama,M., Teranishi, Y. & Nakanishi, S. (2003) Reversible suppression ofglutamatergic neurotransmission of cerebellar granule cells in vivo bygenetically manipulated expression of tetanus neurotoxin light chain.J. Neurosci., 23, 6759–6767.



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Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats