The Spanish Journalol Psychology Copyright 1998by The SpaiúshJourual of Psychology[998,Vol. 1. No, 1,39-58 1138-7416

Early CannabinoidExposureas a Sourceof Vulnerability toOpiateAddiction: A Model in LaboratoryRodents

Miguel Navarro* and FernandoRodríguezde FonsecaComplutenseUniversity of Madrid

Recentfindings haveidentified anendogcnousbrain system mediating ihe actionsofcannab/s sol/ro preparations.This systemincludesIhe brain cannabinoidreceptor(GB-1) and iís endogenousligands anandamideaud 2-arachidonoyl-glycerol.The endogenouscannabinoidsystemis not only presentin theadult brain,huí is alsoactiveat early stagesof brain development.Studiesdevelopedat our laboratoryhaverevealedthai maternalexposureto psychoactivecannabinoidresulisin neuro-developmentalalterations.A modelis proposedin which early A9-tetrahydrocannabinol(THC) exposureduring criticaldcvclopmentalperiodsresultsin permaneníalterationsin brainfuoction by either thestimulation of CB-I receptorspresentduring the development,or by the alterationsinmaternalglucocorticoidsecrerion.Thoscalterationswill be revealedin adulthoodafíerchallengeseither with drogs (le. opiates)or with environmentalstressors(le. nove]ty).They wfll include a modifledpatreraof neuro-chemical,endocdne,andbehavioralresponsesthat might leadultimately lo inadaptationandvulnerabilityto opiateabuse.K~.y vvords: mt, cannobino/ds, perinatal exposure, /,ehav/or op/ates, condit/oning, A CTH,corticosterone, vulnerabitiiy

En los ultimos años se ha logrado identificar un sistema endógeno que media las accionesde los principios activos del cannabis sativa. Este sistema está compuesto por el receptorpara cannabinoides cerebral (CB-1>, y sus ligandos endógenos, la anandamida y el 2-araquídonilglicerol. El sistema cannabinoide endógeno está activo en el cerebro adulto,participando también en el desarrollo cerebral. Los estudios desarrollados en nuestrolaboratorio han demostrado que la exposición maternal durante la gestación y la lactanciaa compuestos activos en el receptor CB-1 conducen a alteraciones en el desarrollocerebral. En este trabajo se propone un modelo en el cual la exposición durante periodoscríticos del desarrollo a J9-tetrahydrocannabinol (THC), el principal cannabinoide psicoactivodel cannabis sativa, induce cambios permanentes en la función cerebral, bien mediantela activación de los receptores CB-1 presentes durante edades tempranas del desarrollo,o bien mediante la alteración de la secreción maternal de glucocorticoides. Estasalteraciones se manifiestan en la edad adulta tras la exposición a desafíos adaptativos,como el tratamiento con drogas de abuso (opiáceos> o el estrés medioambiental (novedad,etc.), que inducen respuestas neuroquimicashormonales y comportamentales anómalas,que se manifiestan como una mayor vulnerabilidad a la adicción.Palabras clave: rata, cannabinoides, exposicion perinata¿ conducta, condicionamiento,morfina, glucocorticoides.

This work wasfundedby te Comisión Interministerialde Cienciay Tecnología(CICYT, grantPM 96/0047),theUniversidadComplutensedeMadrid (ProyectoMultidisciplinar PR218/94-5670) and theDelegación del Gobierno para el Plan NacionalSobreDrogas.

The authors wish ro acknowledge thehard work and expert as.sistance of Pilar Rubio, JoséLuis Martín-Calderón, Ignacio del Arco, RaquelGómez, Olivia Maestre, Leticia Escuredo, YolandaMartinand Cristina Meseguer; andrheyalso thank Joséflórez liria for his technical support.They also thank Dr. María A. Villanda and Dr. Raúl M. Muñoz br their generous support in hormonal determinations. The authors are indebíedto the Narional Institute on Drug Abuse (NIDA. USA) for providing A9-THC, and to Raphael Mechoulamn for the gift of HU-2 10.

* Correspondence concerning ihis article should be addressed to Dr. Miguel Navarro, Departamento dc Psicobiología. Facultad dePsicología. Universidad Complutense de Madrid. 28223 - Madrid (Spain). E-ínail: pspscl0@sisucm.es

39

NAVARRO AND RODRÍGUEZDE FONSECA

The use of Cannabis sativa preparaíions(hashish,marijuana)hasa long history, from folkloric useIn ancienítimes, [o Ihe wide~spreadrecreationalusein Ihe twentiethcentury(Mechoulam, 1986). However, it is only within thepast30 years[bat researchershavebegunto understandtbebiological basisof te pharmacologicaleffects of cannabis.Moreover,te last lO yearshavebeencrucial for identifyingte neuralsubstraíesunderlyingcannabisaetionsin the brain(see Table 1, sectionA). SinceIhe seminaldiscoveryof thecannabinoids,the psychoactivecompoundsof cannabis,withthe isolation of A9-tetrahydrocannabinol(THC) as the maincannabinoid(Gaoni& Mechoulam,1964), extraordinaryadvanceshave led ¡o the syntbesis of psychoactivecannabimimeticagents(Dewey, 1986; Mechoulamet al.,1987), andto Ihe identificationof an endogeneussystemmediatingthe neuro-phannacologicalactionsof cannabinoids.This endogenoussystem,Ihe endocannabinoidsysteín, iscomposedof the CE- 1 brain cannabinoidreceptor(Devane,Dysarz,Johnson,Melvin, & Howletí, 1988; Hcrkenhamet

aL, 1990; Matsuda,Lolait, Hrownstein, Young, & Bonner,1990), andseveralbrain constituentsthathavebeenproposedas endogenousIigands for the CB.-¡ receptor,including

Table 1A. Major Advancesin CannabisResearch.B. Contributions

anandamide(Devaneel al., 1992; Di Marzoet al., 1994)aud2-arachydonoylglycerol(Mechoulamet al., 1995; Stella etal., 1997). Surprisingly,the CB-¡ cannabinoidreceptorispresentin brain arcas, including those relevaní for Iherewardingpropertiesof drogsof abuse(Gardner& Lowinson,1991), in greaterabundanceIban mosíotherknownreceptorsfor neurotransmitters(Herkenhamet al., 1990).

Sincetheisolalion of THC, manystudieshaveaddressed[he effectsof this andother active cannabinoidsin brainfuoction (Dewey, 1986). The pharmacologicalprofile of [hecannabinoidsincludesthe acute induction of analgesia,hypothermia,immobility, andcatalepsy(Abood & Martin,1992; Martín-Calderónel al, 1998), aswell asneuro-endociine

alterations,including the activation of Ihe pituitary adrenalaxis and the inhibition of the secretionof most anteriorpituitary hormones(Martín-Calderónet al., ¡998; Rodríguezde Fonsecael al., 199Ib, Rodrígue¿de Fonseca,Villanúa,

Muñoz, San-Martín-Clarke,& Navarro, 1 995b). Cannabinoidsalso activatemonoaminergicsystemsin the twain, especiallyreward-relatedsystems(Garduer& Lowinson, 1991; Molina-Holgado, Fi Molina-Holgado,E., Leret,González,& Reader,1993: Navarroct al., 1993a, 1993b,in press;Rodríguezde

of the ResearchTrendsdevelopedsince 1993at the Instituto

Universitario de Drogodependencias,Departamentode Psicobiología,Facultad de Psicología, UniversidadComplutensede Madrid.

EVENT REFERENCE

A.

MEDICAL USES OF CANNABIS

ISOLATION OF PSYCHOACT¡VECANNABINOIDS

SYNTHETIC CANNABINOIDS

DISCOVERY OF CANNABINOID RECEPTORS~CB-l)

CLONINO OF CB-¡

ISOLATION, BIOCHEMISflY, & ROLES OF ANANDAMIDE

SYNTHESISOF CE-I ANTAGONIST

ISOLATION & ROLES OF 2-ARA-G

DESCRIPTIONOF UPTAKE SYSTEMS

3000 B.C. TraditionalAsiaíic Medicine. Mechou¡amet al., ¡986

GaoniandMcchoulan,,¡964

Mechoularnel al., ¡987

Devaneet al., ¡988: Herkenhamel al., ¡990

Malsudael al,, ¡990

Devaneel al., 2992;FrideandMechoulan,,¡993;Di Marzo el al., 1994

Rinaldi-Carmonaet al., ¡994

Mechoularnet al., ¡995; Siellaet al., ¡997

Beltramo el al., ¡997

B.

BEHAVIORAL TERATOLOGY OF CANNABINOIDS Navarroel al., 1994ab,1995, 1996; Rubio et al,, 1995, 1998

CANNABINOIDS & OOPAMJNERGICSYSTEMS

NEUROENDOCRINOLOGYOF CANNABINOIDS

CANNABINOIDS, STRESS,& ANXIETY

Navarroet al. ¡998; Rodriguezde Fonsecael al., 1994aandb

Martín-Calderónel al., 1998; Rodríguezde Fonsecaet al., ¡995

Navarroet al., [997;RodríguezdeFonsecael al., 1996

40

NEUROBIOLOGY OF CANNABINOID ADDICTIONiDEPENDENCE Rodríguezdc Fonsecael al., 1997

CANNABIS AND ADDICTION VULNERABILITY 41

Fonsecaet al., 1 992b;Rodríguezde Fonseca,Martín-Calderón,Mechoulam, & Navarro, 1 994a), and induce anxiety-likebehaviorsin laboratoryanimaisandhurnans(Dewey,Gardner& Lowinson, 1991; Halikas, Weller, Mouse, & Hoffman,1985; Navarro et al., 1993a;Rodríguezde Fonsecael al.,1996). Most of theseeffects can be elicited after acuteadministrationof anandamide(Fernández-Ruizet al., 1997;Fride & Mechoulam, ¡993; Romeroet al., 1996) althoughwith someexceptions(Fride e[ al., ¡995).Additionally, therecentsynthesisof thefirst cannabinoidreceptorantagonist,SR 141716A(Rinaldi-Carrnonaet al., 1994)hascontributedto establishthe existenceof anendogenouscannabinoidtone,regulatingboth motor activity and emotional responses(Gueudet,Santucci,Rinaldi-Carmona,Soubrié, & Le Fur,

1995; Navarroet al., ¡997)andhas provided a tool withwhich to unmaskdic neuro-adaptationsunderlyingchronic

cannabisexposure(Rodríguezde Fonseca, Carrera,Navarro,Koob, & Wciss, 1997).

The investigationof the effectsof maternalexposureto

cannabinoidson brain developmenthasbeena very activefield of researchin the cannabinoidfield. Animal researchhas revealed the existence of long-term bebavioralconsequencesof maternalexposureto drugsof abuseduringgestation and lactation (Navarro,Rodríguezde Fonseca,Hernández,Ramos,& Fernández-Ruiz,1994a; Navarro,Rubio, & Rodríguezde Fonseca,1994b;Robins& MilIs,¡993; Zuckerman, ¡991). However,knowledgeof tbe realimpact of maternalexposureto cannabison the developmentandadult expressionof cognitive and behavioralfunctionsin humansis far from being achieved. Cannabissativapreparationsremainthe mostwidely usedillicit drugsduringpregnancyin wes[em countries(Abel, 1980; Dayet al., 1994;Fried, 1995).Cannabinoidscan betransferredfrom the rnotherto theoffspring through placentalblood during gestation(Hutchings,Martin, Gamagaris,Miller, & Fico, ¡989) andthroughmaternalmilk during lactation (Jakubovic,Hattori,

& Mc Geer, ¡977). Thepresenceof psychoactivecannabinoidsin thedevelopingbrain might interfereas epigeneticfactorswith the rigidly ordereddevelopmentalprogramthaI occursduring te oníogenyof the centralnervoussystem,resultingin neuro-developmentalalterations(Mirmiran & Swaab,¡987).Early laboratorystudieshad sbownthat maternalexposure

to either cannabisextractsor THC resultedin ¡ong-lastingalterationsin brain developmentandfunction (Abel; Brake,l-lutchings,Morgan,Lasalle,& Mt, 1987; Dalterio & Bartke,1979; Fernández-Ruiz,Rodríguezde Fonseca,Navarro, &Ramos, ¡992; Hutchings,Morgan, Erake,Shi, & Lasalle,1987; Rodríguezde Fonseca,Cebeira,Fernández-Ruiz,Navarro,& Ramos, ¡99la; Rodríguezde Fonseca,Cebeira,Hernández,Ramos,& Fernández-Ruiz,1990;RodríguezdeFonseca,Hernández,de Miguel, Fernández-Ruiz,& Ramos,1992a;Walters & Carr, 1986).Subsequentstudiesdevelopedin our laboratory(Table 1, referencesin sectionE) revealedthat maternalexposureto cannabinoidsresultedin behavioralalterationswhichcould beobservedeitherduring development

or at adult ages.Ihesealterationswere associatedwith neuro-chemical disturbancesin nionoaminergicand neuro-pepúdesystems(Bonninet al., 1994; Molina-Holgadoet al., 1993;Rubioet al., 1995).

An unexploredaspectof the behavioralteratology ofabuseddrugsis teir possibleroleas avulnerability factorfordrug-seekingbehaviorin theadult. Severalstudieshaveshown

that maternalexposureto opiates(Gagin, Kook, Cohen,&Savil, 1997)or to psychostirnulants(Keller, Lefevre, Raucci,Caríson,& Glick, ¡996) resultedin a sensitizationto thereinforcing effectsof thesecompoundsin theadult offspring,suggestingte establishmentof a vulnerability as a resultofthat earlyexposure.The neurobiologicalmediatorsfor theestablishmentof suchvulnerability remainstobe de[ermined.However, tbepioneerwork of McEwenandcolleagues(1987,and referencestherein) hasestablishedthat underlyingsuchaltera[ions may be the drug exposure-inducedeffects onmaternalsteroid-hormonesecretionsor the intrinsic activityof suchdrugs, which acías steroidhormone-likesubstances.Most dmgsof abusearepotentactivatorsof te hypothalamo-

pituitary-adrena¡axis (HPA), aud it hasbeenproposedthatmaternalstressinducedby Ihese drugsmigbt underlie [heobservedbehavioralsensitization(Molina, Wagner,& Spear,1994; Rubio et al., 1995). The effects of suchepigeneticintIuencesin brain rewardsystemsbavebeenstudiedby theexíensiveresearchof Piazza’sgroup ( Piazza,Deminiere,LeMoal, & Simon, 1989;Piazza,Deroche,Derniniére,Maccari,Le Moal, & Simon, 1993; Piazza& Le Moal, 1996).Theseauthorshavesuggestedthat both prenatalstressandprenataldrugexposureseemto play an importanírole in te individualpredisposition[o psychostimulantself-administrationin rodents,by the induction of long-term changesin the activity ofmesocorticolimbic-projectingdopamineneuronscontainingglucocorticoidreceptors(Harfstrandet al., 1986), andHPA

activity in te adu¡toffspring(Callaghanet al, 1994; Deminiéreet al., 1992;Macean et al., 1991). In this regard,tere is littleinformation aboutte possiblelong-termvulnerability-inducingeffectsof maternalexposureto marijuanaor its psychoactivecompounds.Natural cannabinoids,sucb as THC are potentactivatorsof the HPA axis by meansof its interaction withhypo[halamic brain cannabinoidreceptors(RodríguezdeFonseca et al., 1991b,1995b). Perinatal exposure tocannabinoidsmightten resultiii increasedmaternalcirculating¡evels of corticosteroneand could iníerfere with thedevelopmentof te HPA axis in te fetuses. ‘[he fact diaLbrain cannabinoidrecep[ors arepresentin earlystagesofdevelopment(Rodríguezde Fonseca,Ramos,Bonnin, &Fernández-Ruiz,¡993) adds anotherimportantbiologicalsubstrateto the actionsof maternally-deliveredcannabinoidson braindevelopment.

The presentstudy was designedto further analyzetheeffects of maternalexposureto THC in the reinforcingpropertiesof morphinein the adult offspring. Previousstudieshaveshown that endogenousopioid systems(peptidesandteir receptors)canbe alteredafter maternalexposureeither

42 NAVARRO AND RODRíGUEZ DE FONSECA

to stress(Insel, Kinsley, Mann, & Rridges, 1990; Keshet&Weinstock, 1995) or to perinatal cannabinoidtreatments(Kumar et al., ¡990). Becauseof the growing evidencethatthc functional statusof tbe HPA-axis rnight be relevantforopiate-seekingbehaviorin the rat (Saham& Stewart,1995),

sensitivity to the reinforcingpropertiesof a moderaledoseof morphine (350 pglkg, which inducesplace-condi[ioningin only 50% of the control anirnals) was studied in adultoffspring born of mothersexposedduring gestationandlactation [o severaldosesof TI-IC thaI were closely relatad

to humanconsump[ion. ‘[he functional statusof tbe HEA axiswas evaluatedby rneasuringplasmalevels of ACTI-l andcorticosteronein basal condi[ions, andafter the adap[ivechallengeof conditionedplace-preference(CPP) testing.Anotherstress-re¡atedhormone,prolactin (PRL), was alsocontrolled. Since increasedsensitivity lo [be reinforcingeffectsof abuseddrugsbas beenassociatedwith changesin

the pattcrnof locomotorandexploratoryactivity (Piazzactal.. 1989),we also evaluatedthe behavioralresponse[o novelaud familiar environments,using a batteryof behavioraltests.Lastly, in orderto further evaluatethe estab¡isbmentof long-lasting functional alterations as a result oF maternalcannabinoidexposure,we studiedihe effects of maícrnalexposureto the highly potentcannabinoid,(-)- ¡ ¡ -hydroxy-

d-tetrahydrocannabinol-dimethylhepíyl(HU-210), on Iheadult sensitivity of the HPA axis to an acutecannabinoidchallenge.

Method

Suh¡eets

Femalevirgin rats of the Wistar strain (> 8 weeks oíd;2(E-250g) werehousedin aroom with conn’olledphotopexiod

(¡igbts on: 08:00-20:00)and temperature(23 ± lcr). Thcyhad free accessto s[andard food (Panlab,Barcelona)andwaterDaily vaginal smearswere taken between10:00-12:00b, andonly thoseanirnalsexhibiting Ibree or more consistent4-daycycleswere usedin this study. Femalesin theproestrusphasewere allowed to stay with a male for ma[ing, and anew vaginal smearwastakenon tbe next day. Thoseanimalsshowing (he presenceof spermcelis were acceptedasprobably pregnaníandusedfor jt tetrahydrocannabinolorHU-2l0 exposurestudies.‘[be day on which spermplugswere found wasdesignatedthe firsí dayof gestation.After

weaningthe offspring, they were separatedand housed,4-5animalsof thesamesexandtreatmentpercage.Thirty litterswereusedfor the behavioralstudies,distributedas follows:8 vehicle, 4 THC ¡ mg/kg, 4 THC 5 mg/kg. 3 THC 20 mg/kg,4 HU-210 1 pglkg, 4 HU-210 5 pglkg, and 3 HU-2¡O 25¡iglkg. Por both CPPand defensive-withdrawalstudies,2-3maleoffspring aral 2-3 femaleoffspringpor litter were chosenrandomlyat adulí age(>70 days). Femaleratswere studiedin the estrouspbaseof the cycle.

Cannulae implantation. For [he ti me courseof theendocrineac[ions of HU-210, a group of six animalsportrcalrnentwas implantedunderEquithesinanesthesiawithindwelling atrial cannulae,insertedvia the rigbt externaljugular vein. Cannulaewere filled witb hepariniLedsalme

(lO U/mi) lo maintain patency.AnimaN were allowed alleasí48 hours¡br rccovery,prior to experimentalprocedures.

AII the procedureswere carried out accordingto theFuropeanCnmmunitiesCouncil directive of 24 November,¡986, (86/6<19/EEC)rcgulatinganimal research.

Experiments

In the t’irst experiment,we studiedthe performanceontheelevatedplus-mazeof maje andfemaleoffspring who hadundergonethediffcrent perinatal[reatmentswi[h TI-IC during

pos[natal development.‘[he animalswere rcpeatcdlyexposedto themazeal 2<), 30, 40, and70 days of pos[natal life.

[u lEe seceudexperirnení,adulí animaisof both sexos(>70 days). born of mothers exposed lo Ihe differentexperimentaltrea[ments. were studicd in Ihe defensivo-withdrawa¡ test under novelty conditions (RodríguezdeFonsecaet al., 1996). Ono wcck after thetesting procedure,íhey were divided into two groups. Rats of the first groupwerc left undisturbodfor íwo addi[iona¡ wcoks. ‘[he animalswere killed aftorbabituatingIhein tu Iho bandlingprocedure.audplasmasamploswere obíained(basalgroup). Thesecondgroupwasused to síudy tho effects of perinatalexposure[o

THC un the reinforcing propertiesof a moderatedoseofmorphine (350 pg/kg, Rubio et al. ¡995), using the CPP

paradigm. Locomotor aclivity was measured duringconditiuningsessions.Ihesoanimaiswere killed afíer Ibeendof the 45-minutetosting sessiunof the CPP tost, andplasmasamplosworecollected(placo-proferencegroup).

In the third experiment.animalsexpusedtu (-)-l 1-hydroxy-AtTetrahydrocannabinol-dimethylheptyl(HU-210)wero usod for evaluatingthe time-courseof the adrenal

responselo an aculecannahinoidchallenge.Adulí animalsfrom Ihe differen[ gruups (u = 6; > 70 days) bearingindwelling atrial cannulacwore injoctedwith I-IU-210 (20pglkg, iv), aud plasmasampleswere obtainedfrom Ihejugulce vein -20,0.30,60,aud 120minutesbefureaudaftaIhe injection of [he cannabinoidagonisí.

Drugs ¿vid Treatments

Fermata!cannabinoidexposure.A9-tetrahydrocannabinol(THC) of greaterthan 95% purity was provided in an ethanolsolution by dic NationalInstituto on DmgAbuse(Projecí 488&OB). (-)-I 1 ~hydroxy~fl&tetrahydrocannabinol~dimethylbepty¡(HU-210)HU-210wasagil by Dr. RaphaelMechou¡am,from‘[be Hebrew University at Jemsalem.It was dissu¡vedin 95%elbanol al a concenuationof lO mg/kg. lmmediately before

CANNABIS AND ADUICTION VULNERABILITY 43

use,te etanol wasevaporaredandte residuowasomulsifiod

using sosameoil as the vohiclo. Pregnanífemalosreceivedadaily singleoral duseof THC (1, Sur20 mg/kg budywoight,given betwoon10:00 and ¡2:00 am.), HU-210 (1,5 ur 25

vg/kg) ur vehicle in avolurno uf 0.1 ml. ‘[he treatmentsíartedin the fifth day of gestationandwasmaintaineduntil the 24t11dayafíerbirth, Iho day un which pups wereweanod.Thedososof THC chosenwero an exírapolatiunfrum curron[ ostimalesof moderatotu heavyexpusurotu Ihis cornpuundin humans,and were corroctod.consideringIho differencosin route ofadministrationandbudy surthcoarea(Rusenkrantz.Spraguc,Floischman,& Braudo, ¡975). Wc have proviuuslyubservedthaI this dusagorosultedin plasmaTHC lovols wiihin tbo rangoof thusoreponedcausingbehavioraland physiulugicaleffec[sin animal modols (Navarroet al., 1993b;Rodríguezde Fonsecaet al., 1991b). I-IU-210 doseswero soloctodun tho basisof itsoquipotencywith ‘[HC (Rodríguezde Fonsecaot al. 1995b,1996).Tu assosstho pussib¡ctuxic effects of the trealmoníproviously reponed(Brake. Hutchings,Murgan,Lasalle, &

Shi, 1987; Huíchingscí al., >987), sovoralgosialionalandlactationalparamo[ers werocuntrolled,as describodelsewbore(Navarroel al., 1994b).Figuro 1 depicísthe lack uf consistent

effectsof matemalexpusuretu THC on fund- andwater-intakeduring gestationandlactation.A similar profile wasobservodaherHU-210 exposuro.

Morphine treatment.Murphino hydrochloridewasusodfor the CPPstudios. It was suppliedby CentroNacional doEstupefacientesy Psicótropos.proparoddaily using isotonicsalino as vohicle, andinjocled intrapcri[oneally al [he dososdescribedin Ihe ExperimonísSoction,in a volume of 0.3ml. Nonoof the morphine dosestosted inducedpbysical

dependence.as ovalualedusing naloxone(1 mg/kg) aflerIhe 3-day cunditioningsessions(Rodríguezdo Fonsecaelal., 1995a).

AcuteHU-210 exposure.HU-210-induceda¡tera[iuns in

Ihe secrotionof corticusleroneandprolaclin wore studiedin animalsboaringindwolling atrial cannulae.HU-210 waspreparodin saline/prupylene-glycol/Tween80 vehiclo (90:5:5y/y), and injectedintravenouslyal a duso of 20 ¡.iglkg in avulumeuf ¡00 pl.

BehavioralTesting

Elevatedplus-mare.The performanceof Ihe uffspringin the elovaled plus-mazowas repeatodlyevaluatodatpostnataldays 20,30,40, and 70, fullowing previuuslydescribedrnotbuds (Rubio el al. 1995). The rosulís woroexprossedas 1) tho absolutotime spontin Ibe exposedarmsuf Ihe mazo,and2) [he number of arm entries. AII ibo

bohavioral studies look placeduring the murning limo oftho light cycle (09:30 - 13:00 br)

Defensivewithdrawal. Defensive-withdrawaltesí wascunductedas previouslydescribod(Rodríguezde Fonsecaeíal., ¡996). Tho apparatusconsis[ed uf an opaqueopon field

(¡00 x lOO x 40cm), the fluor of which was markedwith20 x 20 cm squaros.‘[he field containoda cylindrical

polyethylonochamber,measuring17cmdeepand 10cmindiameten‘[he chamberwas upen al uno end arel si[uatedalongsideIho wall runninglengthwisoand20 cm awayfruma comerof Iho opon tield. me upen tiold was illuminatedíísing a 500W halogoncoiling lighm. which wasrogulalodloyield 350 lux al Ihe centorof iho upen fiold. Tosling was

cunductodonly undernovelty conditiuns.as descrihedin IheExperimcntsSection.‘[he ratswore placedinside iho chamber.

in Ibo opon field, audthe fullowing bohaviorswcre scorodby trainod uhsorvers, who were blind tu experimentalcundi[iuns: ¡atency in leaving Iho chamber(emergence

laíency), definod as placemeníof alí fuur paws in Ihe upenfleld; thc tulal time spenlin Ihe chambor;tho meanlime spentin te chamber(i.e.. the tolal time spont in ihe chamber

divided hy the total numboruf entrios);motoractivity, definedas tho total numberof ¡mes un the fluor of Ihe open ficídcrusseduuísido te chambor(crussings>,andiho numheruf

rearirigsperformedculsido the chamber.‘[he Cosí length was¡5 mm. Ahertesting each animal,Ihe apparaluswascleanedwitb a woak acid solulion (1%. acotic acid) tu preventolfacloiycuesfrum affectingIho behavioruf subsequenllytesíedrats.

Conditioning. Morphino-inducodplaco preforence(CPP)studies wero performedas proviuuslydoscribed(Rodríguez

do Fonsecaci al., 1995a),usinga throe-armapparatussimilartu Ihal descrihedby Hand, Slinus, & Le Moal (¡989). ‘[he

apparatuscunsistoduf throo intercunnoctedrectangularboxes.measunng40 x 35 x 35 cm, situatodat 12(1’ anglosfrom eachuther. In ihe middle, therewasa triangularareawiíh a smoothglass Iluor, Irum which anyuf the threo cumpartmentswereaccessible.Eachcumpartmentwasequippedwith a differenl

setof sensurysíimuli: compartmeníA wasequippedwilh asandtluor. plain walls, anda small containorwiíh a drup uf¡0% aceticacid. ComparímeníE cuntainodaromuvablesufíplaslic fluor, walls painted wiíh white dut circlos (7.5 cm),

anda small cuntainerwith a drup of anisooxtract. Finally,compartmen[ C hada cork tioui; altornatingwhito strips (5clii wide) paintedun ¡he walls. andno odur (a containerwithdistillod walor). ‘[he apparatuswas placed in a dimlyilluminated (11<) Lux) isolatedroom. Eachcumpartmenlwasequippedwith eighl photucollswhich allowed us [u monitortho pusition of Ihe animal,andlo autumaticallyregistorthetimo spenl in cacbcompartmenl.Afler lesling eachanimal,the floors werechangedai~d washodlo avuid odurcues.EachCPP experimenícunsistodof a5-day sehodule,with íhreephases:preconditioning,conditiuning andlesting. Animalsexhibitingstrong unconditiunedavorsions(< ¡0% of thosessiun)or preferencos(> 60% uf Ihe session)for anycompartmonlin the45-minutoprecondiíioningsessionwerediscardedfor the conditioning procedures.‘[huso lwocumpartmenlstu whicb Ihe animalsexhibitedte musísimilarpreference-timewerorandumlyassignedfor ihe cunditioningprocedure.This cunsisteduf a 3-day seheduloof doublecundilioning sessions.The first day invulveda momning session

44 NAVARRO ANO RODRíGUEZDF. FONSECA

(9:00 - 13:00) in wbich animalsreceiveda singleduseufmorphine(a fulí duso-rospunse(experimení 1 ( or 350 g/kgip, in THC-exposodanimals)and were immediaíely placedin onoof Ihe cumpartrnents.During Ihis 30-mm cunditioningsession,tho animalswere not a¡lowedtu explore Ihe other

compartmentsuf te apparatus.In [he eveningsession(16:00- 19:00) thoanimalsreceiveda singleintraperitunoalinjeclionuf salinosolution, and were placedfor 30 mm in the othercornpartmentchosonfor conditioning.On Iho seconddayofcunditioning, Iho ratsreceivedthesalino injoctions in [hemorningsessiunand [he drugadministrationin Iho oveningsession.OnIhe third dayuf cunditiuning,[be samesohodulowasusod as un Ihe first une. On thebasisuf preliminarygludios in unir laboratury,we chuseIhis schedute[o avoidcircadianvariability (morning/evening).Aftor threedaysufconditiuning,[he animalswere againallowedtu freely exploroIhe lhree compartmonts,as in [he precunditioningphase

(testingsession).The absolutotime spentin eachcumpartmentwasautomaíicallyregisterodandusedfur Iho evaluatiunof[heCPP.

Locomotoractivity. During the different conditiuningsossions,[he pusitiun of Ihe animal in the cumpartmentwasautumaticallyregisteredby meansof the photocellssystomdescribedaboye,andIbe tutal numborof beaminterruptionswasusedfor tho analysisof locomotorac[ivity (Rodríguez

de Fonsecaet al. 1 995a).

Samplingand Hormonal Deíerminations

Throo hundredmicroliters of bluod wore withdrawnat-20,0,30, 60, and 120 minutesboforoandafter Ihe injection

uf HU-210 from [hejugular vein witb a heparinizodsyringe.Sixly minutesafter ub[aining Iho lasí samplo,tho animalswere saerificedby rapid decapitationusing a guillotine.

Animais werepreviously farniliarized with this handlingproceduro.Trunk bluod was collected in tuboscontaining400 ¡tI of 6% EDTA, and centrifugedat 2500 x g at 40C.Plasmawas stored, frozon al -200C, until assayedfurhormonaldeterminatiuns.Plasmacurticosteronolevels weremeasuredby a radioimmunu-assaysystem(RIA), usingaspeciflcantibodyfrom Bio Clin (Cardiff, Walos). This RíA-systernyiolds basal valuesof corticosteroneof ¶75 ±25ng/ml in undisturbodadult malo animals,and 500 ±70 ng/mlin stressodanimals(Rodríguezde Fonsecaet al., 1996). ‘[hevariability uf the methodwas ¡5.3%,and[he detectionlimitwas 62 pg/ml. PlasmaPRL levels wero moasured,aspreviouslydescribed(Rodríguezde Fonsecaet al., 1995b),by a spocific doub¡e-antibudyRIA-system,using ma[eria¡skindly providedby [he Natiunal HormuneandPituitaryProgram(NIH, Belbesda,MD, USA). Valuesaro expressedin ng/ml of roferencepreparalionrPRL-RP3.Tho intra-assaycuefficient of variationwas 3.3% and Ihe sensitivity was0.025 ng/ml. AlI sainplosweromeasuredin the sameassaytu avoid interassayvariations.

StatistiealAnalyses

Twu levels uf analysiswere performedin Iho presenístudy. Dala of individual animals wero assessedbymul[ifaclorial analysisuf variance,asrequired. Fullowing a

signifocantE value,posthoc analyses(Newman-Kouls)wereperformedfur assessingspecifocgruupcomparisuns.Lilteranalysoswore perfurmodfollowing 1-lolsun ciad Pearce(¡992)suggestions,in ordertu assess[he presenceuf lilter effects.‘[o this end,ANOVAs wereperfurmedusing the meanlitíervalue ubservodasIhe unit lur analysis.AII calculaíionswere

performedusingthe BMDP statisticalpackage.

Rosults

MATERNAL FOOD INTAKEDURING GESTATION AND LACTATION

70

60

o,

ooou-

30

20

lo

MATERNAF. WATER INTAKEDURING GESTArION ANO LACTATION

100

uJ6— 40

20

EXPERIMENTAL DAY

figure 1. Lack of consisteoreffocísof maternalexpusuretu THC

(0, 1, 5 or 20 mg/kg} un ibud- and water-intakeduringgestationandlaclalion. Ordinalesaremeansof the daily inlake of oitherstandardfood pelletsnr tapwater. (*) p < 0.05, Newman-Keuls,versus vehicle- treatodanimals.

~TkERW~~~T

50 —g-—— VsHIaE—•--— NC 1.—Q.—— 1-J.Ic5

40 -.-—.‘.—— THC2C

suTIl

0

OESTATK»~ LACTATION

EXPERIMENTAL DM

.-e— VEIICLE—.--— INC 1

—O—— 7H05

eo —e—— TI-1020

BIRIH

OSETAIlON LACTATION

CANNABIS AND ADDICTION VULNERABILITY 45

Efjécts of Perinatal Exposureto THC on SeveralGesitational andLactational Parameters

In order lo assossthe possibleloxie and nutritional effectsuf ‘[HG administratiun, repurtedelsewhere(Brake et al.,1987;Hutchingset al., 1987),we recordedseveralparametorsthroughuutIhe gosíationand lactatiun (seo Figure 1), aspreviously described(Navarroet al., 1994b).Our resultsshuwedthat treatmentwith ‘[HC 20 mg/kg reducedmaternal

food-intakeduring the first day of treaímenr(simple offectuf dose),F(3, 12) = 7A>p < .005, and Ihesecund,F(3, 12)

eO2.4

owOoo.ww1-

z1’-2wo.faw2

120 -

100 -

80 -

60 -

40 -

20 -

O

MALES *

*

= 8.7, p < .003, disappearingthereafter.Moreover, overalíANOVA analysisdid nol revealdifferencosin matemalfuod-

intake throughouit the entire gestation,1(3, 12) = 1.2, p =

.35 (nonsignificaní).Mothersexposedtu THC 1 mg/kg drankmore wator during gestation,F(3, 12) = 4.6, p < .02,althuughthis effect disappoarodduringthe lactational poriod.

Perinatal‘[HG exposuredid nul result in differenceseitherin maternalweigbt-gain, or in Ihe size and weight of tho

lilíers (data not shuwn). Weigbt gain of the offspring,measurodun postnataldays lO, 15, 20, 30, and40 wasequalin all Ihe experimentalgruups(datanot shown).

e02

.4

ola0oo.la

la

5-

2

5-2lao.fa

la2

20 30 40 70

POSTNATAL DAY

20

u>la

1-2la

24

-J.41~-o1-

16

10

5

o

20O>wE1-2la

2

< 10-I.41-ou-

o

100FEMALES

* *

80 160

40 1

20

O20 30 40

POSTNATAL DAY

OcaL2 THC~••

Figure 2. Developmentalporformancoin Ihe elevatedplus-mazoof maje andfomaleuffspring perinatallyexposodlo THC (0, 1,5 or20 mg/kg). Upporpanel,Urnespent in Iho oxposedarms;lower panels, total arm entiles,Valuesare means±SFM. of 8-12doterminations

por group. (*) p =<1.05, Newman- Keuls, versusvehicle-trealedanimaN.

70

20 30 40 70

POSTNATAL DAY

20 30 40 70

POSTNATAL DAY

NAVARRO AND RODRÍGUEZl)E FONSECA

Effects of Maternal Exposuretu THC un BehaviuralPe4bnnanceiv the EIeí’ated Plus-Muzí’

The analysisuf thc perfurtuancein Ihe elevatedplus-mazerovoaled adoveloprnenlalcffrct boíh in thetimo spentlii the exposodarmsant> in dio nuniheruf arm entiles. 1<3.303) = ¡2.7, p < .0001, andF(3. 3<13) = 22.2. ,‘, < .0001.respectively.with a markedlydifíerení responsein Ihe adulíage(postnataldey 70). Both otfectsappoarcdas a rcsult ufmaternaloxposurolo THC: main effectof maternaltrcatrnent,1(9, 303) = ¡.9, p < .05; and [‘(9, 3031 = 3.6, p < <105.frr time speníuuí and arm erarios rospecíively. flowevor,the nalurouf Iho alterationsin expluratury and locomotorbehaviurs soemodtu be differont. Thus, a clear sex xtrealmentx pustnatal—dayinícíaclionappearedin Ihe analysisuf Ibe lime spen¡ il] dio exposedarrasof Iho mazo.149,303)

0

ti2S.J5-4-J

wuzu,<5

S.J2w

500

500

400

300

200

loo

o

400

300

015zu>u>o(3

200

100

o

3,9, ,o < .01: malo ollspring uf muthersexpusedtu cilberTiIC 1 ur 5 mg/kg oxhibited bigherexpluralurybobavions.whereasIhuseexposodtu 11 IC 20 mg/kg exhibitedaconstanítondoncy lo avoid ihe explurationof tho expusedarmsuf

[he mase.Femalooffspring also oxhihitedchangosin thepanerou> expiuretionu! Ihe upenanosun pusínataldcv 70.altbuughthe changoswere 1101 dose-dependont,appearingal‘[lIC ¡ and 20 mg/kg. híít no? al 5 mg/kg. Regardinglocomotoractivity in tbe mase,malo offspring nf muthersexposedlo ‘[HG 20 mg/kg exhibitod a significaní decreaso

in thc numberof arm oníriesíhruughoutdevoluptuení.Adulluffspring expusedtu ‘[lIC 1 mg/kg oxhibitod cm upposite

patternun pusínalalday 70. wi íb a high n umbor of armcotries. Femalesdisplaved sume changos tliruughuutdevoluprnent,hui thoy vero nuí consistení.and were notpresea?;.íl adoIt ages.

700

ti,m2.4ozw2F24la2

600

500

400

300

200

100

o

80

60

u>(32

.4wrt

40

20

o

SEXMOTHER TREATMENT

OcaLo•• THC 20

SEX

Figure 3, Bohavioral reactivity tu nuvolry, as measuredfo dio defonsive-withdrawal1051, in malo and femaleanin,alshoraof muthorsexposedtu ‘[lIC <0. 1, 5 ur 20 mg/kg). Ordinatesaremeans± SEM. uf 8-12 determinaliunspor group. (5) p < 0.05, Nowínan-Keuls,versusvohicle-treatedanimaL.

46

MALES FEMALES

SEX

MALES FEMALES

SEX

MALES FEMALES MALES FEWLES

CANNABIS ANO ADUICTION VULNERABILITY

Effects of Exposure tu THC un the Peiformance ofAdult Offspring iv the Defensive-WithdrawalTestunder Novelíy Cunditions

‘[he paííern uf behaviur displayod in the defensivo

withdrawal testwas clearly sex-dimorphic,as reflocted inthe sevoralparamoíersscured:female animalsemergedbefure males, [‘(1, 81) = 23.9, p < .000?.they remainedlesstime inside ihe tubo. [‘(1. SI) = 29.7, p < .0001,andthey exhibited highermotur-activity seoresIban malesforcrossings, [‘(1 Sl) = 69.2, p < .0001,and fui rearings,

UNCONOITIONED SPONTANEOUS LOCOMOTION

‘o.4u,O

u.4laO

Sa,

u>

4LI’0

.4S.JO

100

[‘(1,813=39.7, p <.0001. Maternalexposuretu THC ¡ ur5 mg/kg. buí nul tu 20 mg/kg resulted in a decreasedemergencelaíoncy in malo uffspring: sex x treatmentinteraclion,[‘(3,81) = 3.01, p <.04. Malo offspringofíhe‘[HC 1 mg/kg group remainodless time inside¡hetubo, [‘(1,25) 8.75, p <.01, andíhey also exhibitedalower meantime spentin dio small chamberwhencomparodtu controlanimals. [‘(1, 25) = 5.5?, p < .03 (dalanot shown). A

significanísez x treatmenlinteractionwasalsoobservedinbuth the numberof crossings,[‘(3, 81) = 4.72. p < .005,andrearings,[‘(3, Sí) = 4.2, p < .01. scuredduringIho tesí.

LOCOMOTOR ACTIVITY AFTER MORPHINE

B Ñ~ST

ti, 400

.4u,O

.4u, 200O

‘a,

oOt 7H01 7H05TH020 OL 7H01

MALES raMALES

LOCOMOTOR ACTIVITV AFTER MORPIjINE

Figure 4. Locomotoractiviíy uf malo andfomalesaniínalsperinatallyexposedtu THC (0, 1.5 ur 20 mg/kg) in Ihe placo-cunditioningmasemeasuredin: (A) unírcatedhabituatedanimals:(B) during thetirsí conditiuning sessionwith morphine;and(C) during Ihe lasíconditioningsossionwith murphine.Ordinatesarethenumberof beambreaksregisteredfor 45 mm ± SEM of 8-12 determinaíionsporgroup. (*3 p < 005. Newman-Keuls,versusvehicle-trearedanin,als.

47

0<. 7H01 THC5 104020 0<. 7H01 THCSTH020

MALES FEMALES

OK. 1H01 fl40STHC2O 0<. 10101 TI.CST$C20

MALES FEMALES

48 NAVARRO ANO RODRÍGUEZDE FONSECA

EJfrcts of Maternal Exposureto THC onSpontaneous and Morphine-Assuciated LocomuturActivitv

Maternalexposuretu ‘[HG did not result itt alterationsin sponlaneousunconditionedlocomutionexhibitedby adultanimals,independentlyuf Ihe sex variable. However, theresponsolo morphineubsorvedduring conditioningsessionswas sexualíydimurphic, F(¡, 53) 13 1.2, p< .005, and

was affectedby porinalal expusurotu THC, [‘(15, 265) =

2.4!, p < .05. Animals of Iho vohicle groupdid nut showalteredlocomotion aftermurpbine350gg/kgtroatmentduringconditioning sessiuns<seo Figure 4B and 4C). Huwevor,malo animaisbom from mothersoxpusedtu ‘[HG 5 or 20mg/kg exhibited a differential responsotu ñrst morphinewhen comparedwilh ‘[HG ¡ mg/kg gruup, [‘(3, 53)= 3.7,

p < .05. This differeníial responsocuuld he slill ubsorvedin the íliird conditioning sesgion,un which a sensirizedlocomotorresponsetu murphineappoaredin animaisuf IheTHC 1 mg/kg group,[‘(1, 12)=4.l, p= .05.

EffectsofMaternal Expusuretu TI-fC en MorphinePlace-Prefrrencein Adult Offspring

Maternal expusuretu low dusosuf THC resulted inenhancodsensitivity lo dio reinforcing proporries ufmurphine 350 pg/kg displayedby the adult offspring, as

meastíredin tbe placepreforenceparadigni, [‘(3, ¡24) =

4.4, p < .005.’[he effect wassexuallydimorphic,[‘(1, 124)= 11.1, p < .00]. Malo offspring of Ihe THC ¡ and5mg/kg duso group increasedtheir preferoncefor Ihemorphine-pairedcumpartmentwith respecttu thesalino-

pairedcumparímení,[‘(3, 60) 3.3, p < .03. Only fomaleuffspring of Ihe ‘[HG ¡ mg/kg gruup incroased[hoirproferoncefor Ibo morphine-pairedcompartmontwilh

respocttu the saline-pairodcumparímení:simple offectofmaternaloxposurolo ‘[HG ¡ mg/kg. a, 64) 5.7, p

.03. Neithor cuntrol-offspring flor [huso buin of mothersexpusedtu ‘[tIC 20 mg/kg exhihited adecir proforoncofor11w murphine-pairedcumparímoníwhen cumparedlo [he

saline-pairodune.

CHANGE OF PREFERENCE AFTER CONDITIONING

oor

3,zzoozouwo-

zu)u’

300

200

100

o

*

EJ SALINE-PAIREO

•

*

OIL TIlO 1 THO 5 THG 20 OIL THC1 THC5THC2O

MALES FE MAL ES

Figure 5. Murphine (350 gg/kg)-inducedplaceconditioningexpressedas te percontageof changoovor proconditioninglime values.(*) p < 0.05, Newman-Kouls,morphine-pairedvorsussaline-pairedcumpartment.

CANNABIS AND ADDIC’[ION VULNERAE¡LITY

CORTICOSTERONE VARIATIONSAPTER CONDITIONING TESTING

600

A<o *

w s<o-4.4-. 400.4u>.4•z.4laZ 200

la10~ ——

Bu, Baséo 100 —~0<. T>CI T1105114C20

MALE

u>-J<Ji

<Ji.4.4.4‘o.4o

la

oa2

0<. t1~Cl 1FC5T01020

FEMALES

PROLACTIN VARIATIONS AFTERCONDITIONINO TESTINO

y. S534 + 1O>57y R’S — 0491 g

o

z‘3

300 400 500 600 700

1=-

1400

1=•

1 00

D

y — . 4S5.S1 + 175451 R~2 — 0.191

200 300 400 600 600 700

COFI110051ERONE (ng/mi)CORTICOSTEnONE (ng/mi)

Figure 6. (A). Changesin corticostorunelevels(exposed/basalratio x lOO) assuciatodwith the oxposuretu Ihe condiliuning mazo inadulí malo andfemaloanimalsbornuf moíhersexposedlo THC during gesíationandlactation. (B) Changosin plasma-prolactinleveis(exposed/basalratio x lOO) associatedwith Ihe oxposurolo Iho condilioningmazo in thesameanimals.(C) Pusilivo correlationbotweenplasma-corticostoroneloveis and proforoncofui murphino-pairedeumpartmentin malesuf Ihe THC 1 mg/kg dosogruup, buí nol invohiclo-groupanimals (D). (*) p c 0.05, Newman- Kouls,versas vohicle- Ireatedanimals.

Effects of Maternal Exposure to THC on ¡‘¿asmaHormonal LeveisaJter MorphinePlace-Preference

Ecisal corticosteronelevels (datanot shown)wore sex-dimorphic, F(l, 83) = ¡2.03, p < .001, and they woreclearly affoctod by maternal exposuroto THC: sex xtrecitmentinteraction, [‘(1, 83) = 7.66, p < .01. Maternaloxposuretu ‘[HG resultedin high basalcorticosteronolovolsin fernalooffspring and reducedbasallevels uf Ihis steroidin malo offspring of tho ‘[HG ¡ mg/kg group. Exposuretute place-preferencetost incroased[heleveis of this adrenalsteroid: tesíeffect, F(l, 141)= ¡0.2, p < .005. ‘[he ratio,corticosterone levels (CPP groups)/average basalcorticusteronolevels,displayedmarkedsexualdimorphism,[‘(1, 62) = 16.7, p < .0001, showing a clear offect of

maternaltroatmorot,[‘(3, 62) = 6.8, p < .0005,andci mcirkedtreatmentinteraction,[‘(3, 62) = 12.85, p < .0001,

which revealedthat malo offspringof muthors exposedtuTHC-l mg/kg displayeda higher increasein corticosterone

levois as a resultof the exposurotu the CPP test(Figure 6).‘[his activaloryoffect cissociatedtwith CPPtesting wasnotobservodin malesfrom the low THC-expusuregroupswhenplasmaprolactinwas analyzed.Moreover,it appearedin alífemales,indepondentlyof maternalTI-IC exposure,and inmalesfrom Ihe 20 rng/kg-dosegroup. RegressionanalysisrevealedthaI thechangoof preferencefor Ihe morphine-

paired compartmentcorrelatespositivoly with plasmacorticosteronelevels in malesof the ‘[HG 1 mgAg-groups,[‘(1, 8) 6.75, p <.04, r = 0.49. ‘[his positivo correlationwas not observodin Ihe remainingexperimentalgroups.

49

SO e

0<. THOI T1~5 16026 OIl. 1H04 THCST}C20

MALES FEMALES

‘2zu,E

E

zo

1400

1~~

ea,.

200

a

lOO 200

NAVARRO AND RODRÍGUEZDE FONSFCA

Table 2Liver Analysisof Me Effecrs of Perinatal ExpusureconditionedPlace-Preference(CPP) Croups.

Paramelers

Vehiclo

Mother Trealmení

TI-IC ¡ (mc/kg) THG 5 (mc/kg) THC 20 (me/kg)

Emergeneelatency

Males 438,4±¡52.8 87.9±28.3(*) ¡52.6±36.5 (~) 298.7±¡84.9

Fomales 25,1 ±8.2 26,9±¡3.2 ¡5.3±5.1 14.6±2.9

Changoof preference

Mates 284 ±177.5 715.9±246.2(*3 386 ±50.8 36.8 ±84.4

Fomales -53.5 ±73.6 ¡92.5±¡34.7 79.8 ±22.9 -159 ±79.1

Basal ACTH (pg/ml)

Malos 126.1 ±¡0 296 ±73.7 (~) ¡30.4±¡7.6 ¡14.9±42.4

Fon,a¡es 109.9 ±¡4.2 52.4± 31.6 169.7±64.4 ¡01.1 ±4.9

ACTH post CPP (pg/ml)

Males 464.2 ±34.3(#) 248.6±23.6 (*) 320.1 ±46.5(#) 292.3 ±64.2(#)

Females 367.7 ±93.3(#) 220.9 ±26.2 288.3 ±73 218.0±38.7(#)

BasalCorlicosterono(ng/mí)

Males 221.5 ±63 ¡23.4 ±¡O(~) ¡74.4 ±13.3 271.9±¡LS

Females 339.0 ±73.3 646.1 ±102.9(*) 421.9 ±111.8 394.2±90.5

Curricosteronepusí GPP(ng/mí)

Males 408.5±65.2 525.3±77.9(#) 384.0 ±31.8 397.0 ±84.1

Femalos 413,7±¡00.1 451.5 ±122.7 479.0 ±¡¡0.5 529.5 ±37.2

* p < 0.05, NowmanKeulsversus vohicle-trecitedgruup.# p < 0.05, NewmanKeu¡s versos basalgroupuf Ihe gamotreatment.

Litter Analysisof the EffectsSo TJJC

of Perinatal Exposure

Lilter analysisshowedthaIperinalalexposuretu ‘[HG ¡or 5 mg/kgresulted in asignificantdecreasoin Ibe emorgencelatency andin a tendencytu oxhibil adecroasein tho totaltime spontin the compartmentof te defensive-withdrawaltosí, [‘(3,22) = 3.91, p <.02,and[‘(3,22) = 2.52, p <.08,

respectively.‘[hero wcis markedsexualdimorphismin theporformanceof this íest,as describedaboye,cind perinatalTHC exposurofailed tu inducealterationsin femaleoffspring.‘[he offectsof perinatal lreatmont with ‘[HG 1 mg/kg unmurphine-inducedchangoof placeproferencewere preseníwhen Ihe meanlitter value wasconsideredas Ihe unil for

analysis,[‘(3,19) = 3.8, p < .02 (seoTable2). Tbeseoffectsworo sexuaiíydimorphic, [‘(3,19) = 8.7, p <.008, andwere

signiticcirsí in malesexposedtu ‘[HG ¡ mg/kg.ACTH analysisreveciledIhe presenceuf increasodplasma-ACTHlevois as

aresultofthoCPPtesting,[‘(1,44) = 35.7, p< .0001. ‘[herowasa significaní tosí x treatmentinteraction,[‘(3, 44)= 5.4,p < .004,revecilinga disruptingeffect of maternalexposure

tu ‘[HG ¡ mg/kg un dio patternof AGTI-I ¡evelsdisplayedby both te basalandplace-proferencogroups.Lilter analysisconfirmed Ihe sex-dimorphiceffecls of maternal ‘[HGexpusureun basal corticosteronelovels: sex x trealmoníinteraction,[‘(3, 22) = 4.3, p < .02; and[he differentresponsetu tbeCPPtesí, [‘(3, 44)= 3.8, p e .02, in malesandfemalesas a resulíof dio exposuretu ‘[HG.

50

tu THC. Data are avarage /itter Values ±SEM in basalandpost-

800

iau,zOMIu-Cooo

oo

600

400 —

200 —

o

16.

14 -

ac 8-

-J6-

a.4-

2-

o

51CANNABIS AND ADOICTION VULNERABILITY

EFFECT OF MATERNAL EXPOSURE TO HU-210QN TME QLUCOCORTICOIO RESPONSE TOACUTE CANNABINOID TREATMENT

MOflIFR IIU.2l0 TREATMENT <uflAU~

— VEHUE

—O 1

.—.—o--.— 5

6 25

A

*

e

TIME POST-INJECTION {mIn4

EFFECT OF MATERNAL EXPOSURE TO MU-210QN TI-lE PROLACTIN RESPONSE TO ACUTECANNABINOID TPEATMENT

*

MOTHFR I~u-2’0 TRFATMFNT (.,nAc,1

TIME POST-INJECTION (mm.>

Figure 7 Maternalexposurelo Iho cannabinuidreceptoragonisí 1-IU-210 resultsin alloralloasin Ihe responseof both Ihe pituitary-adrenalaxis (A), andprolactinsocretion(B), tu an acutechallengowilh HU-2¡0 in Iho adulí offspring. (~) p < 0.05, Nowman-Keuls,versusf<rst fracíjon. Standardorrursof Ihe meanworo umitíed tu impruvetheclarily of thegraphs.

EffectsofMaternal Exposuretu HU-210un HPASensitivitytu an acuteCannabinoidChallenge

Maternalexposuretu [he highly poíent cannabinoidreceptoragonisí HU-210 resultedin a permanentlyaltered

sensitivity of the HPA axis tu the activating offects ufcannabinoids:doso effect, F(3, 16) = 3.7, p c .05.Maternaloxposure tu HU-210 resulted in lower mean basal

corticosteronelevels(seoFigure 7A). Afíer acuteinjectionuf HU-210, the animalsbom from mothersof Ibe 1 pg/kg-gruupexhibiteda potentiatedandlong-lastingcorticosteroneresponse,with maximal increasesup tu 500% uver mean

basallevels: time effectF(5, 80) = 6,1 p <.0001.This effectwasnol observedin animalsfrom Ihe 25 ig/kg-dosegroup,which shuweda morosubduedresponseof shorterduratiun:time effect,F(5, 80) = 1.2, p = .14 (nonsignificant). Plasma-prolactinresponsetu HIJ-210in controlanimalswasbiphasic,

with a short activating responsoubsorved30 mm after Iheinjection anda subsequentIong-lasting inhibition, F(5, SO)= ¡5.07 p < .0001 (seoFigure 7B). MaternalexposuretuHU-210 only affectedIhe stimulatory phaseof tho responsetu HU-2lO, again io the lower-dosegroup: absenceof simpleeffect of maternaltreatmentwith HU-210, 1 .sg/kg,un 30-mm fraction. F(5, SO) = 1.3, p = .25 (nonsignificant).

Hu-210 INJECTION

20 0 30 60 120 180

HU.210 INJECTION

—e- VEHaS

5

—0—— 25

20 a 30 60 120 180

52 NAVARRO AND RODRÍGUEZDE FONSECA

Discussion

The prosenístudy showsthat maternaloxposurotu Ihopsychoactiveconstituontuf cannabisTHC resultsin a clustorof behavioralandendocrinoaltorationsin the adulí offspringthat can be rovoaled aflor chaiJongeswith drugs (upiales,cannabinoids)or envirunmontalmodificaíions.‘[bus, maternalexposuretu ‘[HG increasodIhe sensitivity tu Iho roinforcing

prupertiosof a moderatoduseuf murphino in dio adultuffspring, as moasuredin the CPPparadigm.‘[bis sensitivitywas greator in animals oxposedtu low dosesuf Ibiscannabinuid(1 and5 mg/kg), andwasfoundtu be sexually

dimurphic. Place-conditiuninginducod by morphino 350pg/kg iii malo uffspring perinatallyexposedtu ‘ERG ¡ mg/kgwassimilar tu that obtainedin control animalsusing doses3-6 times greater(Rodríguezdo Fonsecaet al., 1995a;Rubio,Rodríguez de Fonseca, Martín-Calderón, del Arco,Bartolomé, Villanda, & Navarro,in press).A recont repurtindicatesthatthis increasedsensitivity is extensivotu oporantresponsesfor opiates,using a model of morphineself-adminisíration(Martin eí al., 1996).Theanimalsoxpusodperinatallytu ‘ERG cilso exhibitedsexuallydimorphicchangosin [he behavioralreactivity lo novel envirunments(seoFigure

3 fur Ihe defonsivo-wiíhdrawalstudy) andin Iho oxploraíorybehaviorsof a familiar onvironmentwiíh aversiveprupertios(seoFigure 2 fur Ihe plus-mazostudy). The endocrinoaltoratiunsobservodincludedsexuallydimorphic alterations

un Iho reactivity of Ibe hypothalamu-piíuitary-adrenalaxislo dio adaptivechallengeof dio CPPtesting.Malo offspringborn of mothorsoxposedtu THC (1 urS mg/kg) displayednormaltu luw basallovels of corticusteruno,andan enhancod

adrenalresponsetu theCPP challenge.Huwover, femaleoffspring perinatcilly exposedtu ‘[HG (1 ur 5 mg/kg)

• displayodthe oppositepattem:permanontlyelevatedplasmalevols of corticosteroneand ablunted adrenalresponsotutbeHPA-activatingeffectsof dio CPP íost. Lastly, malesbum of mothersexpusodlo dio polent cannabinoidHU-210

exhibiteda doso-relatodaltoredHPA responsetu cm acutecannabinoidchallengo:animalsbom frum mothorsexposed

tu [he low doseof thecannabinoid(1 ¡ig\kg) exhibitedasensitizod and long-lasting responsetu acute HU-210exposure,whereasIhosoborn of moíhois treatodwilh Ihohighor doso (25 ~¡g/kg) displayeda desensilizedresponsotu the acutecannabinoidchallengo.Prolactin responsedisplayod sumesubíle a¡íerationsin tho ¡uwor-dosogruup,although tboy only ciffected tho transientactivaloryeffectof cannabinoidin its socretion.‘[he resultaobservedin malesareconsistentwith proviously reportedfindings which roflecí[bar early lito oxpeuiences,sucb asprenatalstressorperinatalexpusurotu opiates,amphotamine,and cocaino, rnightcunstitutoa vulnerabiliíy factor fur drug abuse(Gallaghanel al., 1994; Deminiéro et al., 1992; Gagin el al., 1997;Kollor el al. 1996; Piazza& Le Moal, 1996).

An imbalancein the mesulimbic dupciminorgicnetworkhasbeenconsiderodas tbeneuro-chemicalbasis of the

vulnerability tu psychostimulaníself-administraíionin rodents(Piazzaet al., 1989). As describedaboye,matemalexposurotu cannabinoidsnot only resultsin developmontala¡terations

uf [he dopaminergicsystemsin Ihe brain (Bonnin el al.,1994; Rodríguezdo Fonsecael al., 1990, 1991a, 1992a),

but lii permaneníneuro-chemicalalteratjoasin adulthoud(Garcíaot al. 1996; Navarro, de Miguel, RodríguezdeFonseca, Ramos, & Fernández-Ruiz, 1996; Navarro,Rodríguezde Fonseca,Hernández,Ramos,& Fernández-Ruiz, 1994a; Navarro, Rubio, & Rodríguezde Fonseca,1995;Rubio el al., ¡995). lf we considorthesoobservatiunsíogetherwilh the data reflecting the alíeratiuns in thebohaviural reactivity tu novelty and the responsetumorphine,as well as tho UPA a¡lorations,we can arguethat

maternalexposuretu ‘[HG leadstu a vulnerablephenotype,partially resemblingthaI proposedfui amphotaminesolf-administration: mesucorticolimbicdupaminorgicaltorations,increasodbehaviural responsestu nuvelty, and a sharpadrencilresponsetu adaptivochallengos(Piazzaet al., 1959,1993, 1996). ‘[his propusodmodel uf vulnerabiliíy is depictedin Figure 8. Furtherresearchis needodtu establishtu wbatoxtent it can be also rolevaní for human species.

In Ibo animalsoxpusedtu low dusesof ‘ERG, dio responsotu Iho CPP íost was shiftod tu exhibit buth cm increasedsensitivity lo Ihe roinforcing propertiosof an ED5<> dose ofmurphino (resulting in a necir ¡00% positivo changeofpreferoncein perinatal-exposedanimals),anda clearnseInplasmacuxticosteronelevels, whicbwero positivoly correlalod.

Inlerestingly, prolactinresponsotu tho GP?challengewasnul corrolatedwith dio appearancoof cundilioning,althoughit wasclearly ciffectod by Ihe oxposuretu THG 20 mg/kg..The polent andlong-lasling adronalresponsetu acuteRU-

210 exposurein animalsborn of muthersoxposedtu lowdusosof this cannabinoidis ci remarkablefinding, sincoibistypeof sonsitizedHPA responsehasalsoboen proposodasafactor underlyingvulnorability tu psychustimulantself-adminisíration (Maccari eí al., 1991). Soveralmechanistushayo beenproposedin the elicitation of Iho perinataleffectsof cannabinuids(for reviow, seoFernández-Ruizel al. 1992andNavarro ot al., 1995). ‘[hoy include changosin opioid

peptidesciad thoir recopturs(Kumar el al., ¡990), prenatalstress-likeoffocts (Rubio el aí., 1995), direcí effects undeveíupingmonoaminergiesystems(Bonnin et al., 1994;Navarroel al., 1996; Rodríguezdo Fonsecael al., 1991a;Wal[ers & Can, 1956), andiho activationof brain cannabinoidrecoploiswhich arepresontat birth (Rodríguezde Fonsecael al., 1993). Bosidos tbe proviuusly describodactions ofcannabinoidsun Ihe develuptuontalprofile of dopaminergiecelís,apossiblecannabinuid-ioduceddevelupmeotalalteratiunin opioid peptidesandtbeir receptorsmusí be considorodesa factor underlying Ihe increasedsonsitivity tu morphino-inducedplacepreferonce.It hasboen proviously doscribed

thaI perinatal cannabinoidoxposuro can (a) alter Ihedevelopmeníalexpressionof upioid peplidosin dio reí brain

(Kumaret al., 1990),and(b) inducechangosin upiuid-related

CANNABIS AND ADDIGTION VULNERABILI’[Y 53

EARLY ENVIRONMENTALEVENTS

‘IrMATERNAL

GLUCOCORTICOIDSECRETION

ENVIRONMENTAL CHALLENGESDRUGS (CANNABINOIDS, OPIATES)

STRESS (NOVEL’[Y, SOCIAL...>

ADULT BRAIN

‘1$

‘I~ * *

Figure 8. Proposedmodel of vulnerability tu onvirunmentalchallengosinducedby maternalexpusuretu THC, Ihepyschoactivoconstilueníof Cannabissativa, in rodenís.THG exposureduring crilical dove¡opmenlalporiods may resulí in permanenía¡teraíionsin brainstructuro/funclionby oithor Ihe sílmulationof GB-l roceptorspreseníduring Iho dovolopment,or tho alleraíionsin maternalglucucorticoidsocretions,a known sourceof opigenetie-inducoddovelopmontalplasticily. Adulí challongesoither with drugs(i.e. opiales)or withenvironmentalstrossors(i.e. nuvetty)will result in a modifiedpaííernof neuro-chemical,endocrino,andbohavioralresponses,leadingultimalely lo inadaptionandvulnerabilily.

¡ DEVELOPING BFlAIN 3

ALTEPED NEUROCHEMICAL RESPONSES

MONOAMINES, NEUROPEPTIDES

ALTERES NEUROENDOCIRINIE RESPONSESENHANCED GLUCOCORTICOID SECRETION

ALTERES SEHAVIORAL RESPONSES

ANXIETY-LIKE BEHAVIORSIMPAIRMENT OF LEARNING SKILLS

INDIVIDUAL VULNERABOLITY TO OPUS ASURE:

ADDICTIVE PERSONALITYRISK BEHAVIORS

SENSITIZED RESPONSES TO DRUGS

54 NAVARRO AND RODRÍGUEZDE FONSECA

behaviors(Vela,Fuentes,Bonnin,Fernández-Ruiz,& Ruiz-Gayo, 1995),namely analoxone(5 mg/kg)-inducedopioid-

liko abstinencesyndromoin weanlingmalesexposodtu thocannabinoid,auddove¡upmontalalíeralionsin bo[h painsonsilivity and the analgotie prupertiesuf morphine.Moreuver,íbero is a decir associationbetwoenondogenousopluid andcannabinuidsystemsin reward-relatedaroas,supportingan importantinteractionin the modulatiun by Ihecannabinoidsystemof tho reinforcing propertiesof opicites(Gardner& Luwinson,1991).

The sexualdimorphismalroadyreported,which hasalsobeenobsorvedin tho prosentsludy, is ci communfinding inporinatalcannabinoidstudies(Dalíerio andBaríke, 1979;Fernández-Ruizet al., 1992; Navarroel al., 1994b),andmight be reflocting Iho sex- dopondentdevelopmentalprofileuf bodi opiuid roceptors(Hammer, ¡985), and cannabinoidreceptors(Rodríguezde Fonsecaet ‘al. 1993) in Ihe ral brain.An additiunal explanatiunfor tho effectsdescribedafterperinatalTHG might be a possiblocannabinoid-inducedsúess-likeeffecí. ‘[bis prenatalstress-likoeffectmighí occurthrough a THC-inducedactivationof maternalHPA axis,resulting in a riso of plasma-corticosteronelovels in Ihefetuses,which are dopondentun matornal levols (Ward &Weisz, 1984; seo Figure 5). Howevor,this possibility muststill beconclusively detormined.Reconístudies(McCurmick,Smytho,Sharma,& Moaney, ¡995; Valleé et al., 1997) hayodescribodhow malernal-rostrainístress resultod in apermanontincreaseof basalcorticusteronelevelsin Ihe adultfemale offspring, which is tho samefinding that wo hayoobservedafíer perinatalexposurelo ‘[HG. and which hasaÑo boenfound after perinatalexposuretu alcohol in rals(Lee & Rivier, 1996; ‘[aylur, Branch,Nelson, Fane,& Poland,1986).Additiunally, pronalal sírosshasboen found tu alterIhe upioid contribution tu Iho bohavioral responsotu novelenvironments(Poltyrov & Weinstuck. 1997). In any case,itis worlhy uf note that the offecís of porinalal ‘[HG unmorphino sonsitivityand HPA activity maybe independoní,althuugh associatodin malo animais oxposedtu the luwordoseof THG. It romains tu be determinedwhethor Ihoassociatiunbetweenmorphinoroinforcomen[ and HPA activilyis sexuallydimorphic in naivo animals,ospocially if weconsider that tho studies which hayo set a role forglucucordcoidsun opiMo reinfurcement(seo Saham &Stewart, 1995) hayo beonporformodunly wilh males,Othergonder-rolatodfindings arosofrum the presentstudy: malorats bom of mothersoxposedtu ‘[HG (1 orSmg/kg)displayedmore expluratory behaviorin the defonsive-withdrawalparadigm,which might be consideredtu resemblea femalepatternof behavior.Huwevor, they displayeda groalersensitivity tu the reinforcing proportiesuf morphinothan didfemalesfrom Ihe sameexperimentalgroups.In addition,a¡though females also showod an altered responselo

morphino whencomparedtu controis,lhey did not displayan onhancedadronalresponsetu [beGPPtestingbutexhibitedpermanentlyelovatedbasal levels uf corticosterune.These

dala suggosttha[ perinatal‘[HG did nol resul[ either infeminizaliun nor in masculinizationof thebehavioralandendocrinoparametersstudied.We might concludefrom Ihesefindings that [be proposedrole of [be HPA in [be vulnerabilitytu opiate reinforcoment is soxually dimorphic, andlhatdifforen[ial mechanismsmay be undorlyingibis responsein

both sexos.‘[he surprising officacy of Iho low dusosof THC fur

eliciting long-torm behaviuralaltorcitionsmay be relatedtuIhe well-ducumen[edbiphasicactionsuf’[HG (Dowey, ¡986)and anandamide(Fride el al., 1995),which cummonlyinduceuppusing cictions al low dusos (0.2 -2.0 mg/kg) whencumparedlo higherunes(5-50 mg/kg). ‘[his hasalso boonubservedusinganandamide,Ihe proposedendogenousligand

fur the brain cannabinuidreceptor(Frido & Mechoulam,1996a,1996b; Wonger,Fragkakis,Giannikou,& Yiannakis,1997). In any case,we obsorvedthatuffspringof muthersoxpusodtu ‘[HG 20 mg/kg, adoso tha[ is 10-20timeshighorthanthat estimatodfor humancunsumplionof 2-3 marijuanacigaretiespor day (Rosenkrmrtzel al., ¶975),did nol oxhibiloilher iheincroasedsensitivity tu Ihe reinforcingproporties

of morphinenor tho altorod responsetu novelty. Huwevor,lhey did displciy altoralions in Iho locomotoractivily (seoFigures 2 cind 3), in accurdancewitb proviuus studies(Navarroet al., 1994a, ¡996). Regardlessof ihe lack of

offecís obsorvedin the GPPparadigm,it is importaní turemark that olher mechanisms,such as altoratiunsin theontugony of cannabinoidreceptois,nutritional deficits, orolher adaptivoprucessesmay precludothe appoarancoofIhe effeclsobsorvedwith tho lower dusos.

In surnmary,porinatalexposuretu ‘[HG dosesrelalodtuhumanconsumplionproduceda clear shifí in the sensitivitytu the roinforcingproportiosuf murphinoin malo offspring.lnterestingly, rocenthypothosisatíributetho onsel of drugaddicíion lo a drug-inducedprogrossivedysregulationofhomeosíatieprocossescontrulling behaviural.nouru-chemical,and ondocrineresponsestu relevantexternalstimuli (Kuob& Lo Mocil, ¡997). The factthai cannabisconsumptionrnayresulí in depondonceandaddictiun (Rodríguezde Fonsecael al., 1997)and that a recentroport has linked a particularCB-l receptorgenealbIo tu intravenuususeuf opiatosandpsychostimulants(Gomings ot al., 1997) pruvide furthersuppurltu [benution of cannabinoidexposuroas a putentialvulnerabilily factor fur drug abuso.

Reforences

Abel, EL. (¡980). Prenatalexposuretu cannabis:acritical reviowof offects un gruwth,dovo¡opmont,and bohavior. BehaviouralandNeural Biology, 29, 137-145.

Abood, ME., & Martin, BR. (1992). Nourobiologyuf marijuana

abuse.Trends in Neurosciences,13. 201-206.Boltramo, M., Síella, N., Galignano,A., Lin, S.Y.. Makriyannis,

A., & Piomelli, 171. (1997). Funclional role of high-affinity

CANNABIS AND AUDICTION VULNERABILITY

anandamidetransporí,as rovealedby selective inhibition.

Sejence,277, ¡094-1097.Bonnin,A., deMiguel, R., Rodrfguez-Manzaneque,J.C.,Fernández-

Ruiz, J.J..Santos,A., & Ramos,JA. (1994). Changosintyrosino hydroxylasegene expressiuniii mesencophaíiccatochulaminergioneurunsuf immature andadult malo ralsporinatallyexposedto cannabinoids.DevelopmenralBrainResearch, Si, t47-t53.

Brake.SC., Hulchings, D.F., Morgan,B., Lasalle, E., & Shi, T.

(¡987). Delta-9- letrahydrocannabinolduringpregnancyin Iheral: II Effects un untogony of locomotoractivi[y and nippleatlachnientin Iho uffspring. NeumtoxicologyandTeratology,

9, 45-49Callaghan,PM., Delagarza,R., Cunnighan,K.A., Henry,G., Kabbaj,

M.. Simun,1±,Le Moal, M., & Maccaii, 5. (1994). PrenatalstressincroasesIhe hypothalamo-pituitaiy-adronalaxis responsoin youngandadulí rats. Journalof Neuroendocrinology,6.341- 345.

Comings,DE., Muhíeman,D., Gade,R., Johnson.P., Verde, R.,Sucier, O., & MacMurray, J. (¡997). Cannabinoidreceptorgene(CNRI): assuciadonwith iv. drug use. MolecularPsychiatry,2, 161-168.

Dalterio,5., & Barte,A. (1979).Perinataloxpesuretu cannabinuidsalíersmalo reproductivofuncíiun in mice. Sejence,205, 1420-1422.

Day, NL.. Richardson,CIA., Goldschrnidt,L., Robles.N., Taylor,PM., S[offer, D.S., & Comelius,MD. (1994).Effectof prenatalmarijuanaexposureun [hecugnitivedevelupmentuf uffspringalagothroe.NeurotoxicologyandTerarology, 16, 169-175.

Deminiere,J.M., Piazza,PV., Guegan,G., Abrous,N., Maccari,5., Le Moal, M., & Simon,H. (¡992). Increasedlocomotorresponselo novelty andpropensitytu intravenuusamphetamine

self-administrationin aduil offspringof stressedmolhers.BrainResearch,586, ¡35-139.

Devane,W.A., Dysarz. FA., Johnson.MR., Melvin, LS., &Howlell, A.G. (1988). Determinalionandcharaclerizalionofa cannabinoidreceptorin rat brain. MolecularFharmacology,34, 605-613.

Devane,W.A., Hanus,L., Breuer,A., Portwee,RO., S[ovensun,LA., Orjífin, O., Gibson,D., Mandelbaum,A., Etinger,A., &Mechoulam,R. (1992).. Isulation ands[ruclure uf a brainconstiluenrthat binds tu [he cannabinoidreceptor.Science,258, 1946-1949.

Dewey,W.L. (1986). CannabinoidPharmacu¡ogy.PharmaculogicalReviews38, ¡51-178.

Di Marzo, V., Fontana,A.. Cadas,H., Schinolli, 5., Cimino, O.,Schwarlz, J.C., & Piumelli, D. (1994). Furmaliun andinaetivationof endogenuuscannabinuidanandamidein centralneurons.Nature, 372,686-691.

Fernández-Ruiz,JI.,Muñoz, R.M., Romero,J., Villanda, MA.,Makriyannis, A., & Ramos,JA. (1997). Time-courseuf Iheeffects of different cannabirnimeticsun prulactin andgonado[rophin secretion:evidencefur [hepresenceof CB-lreeeptorsin hypothalamicsírucluresandIheir invulvemeníinIhe effecíof cannahimimeíics.BiochemicalPharmacology53,1919-1927.

Fernández-Ruiz,J.J.,Rodríguezde Fonseca,E, Navarro,M., &Ramos,JA. (¡992). Maternalcannabinoidexpusureandbraindevelopmenl:changosin [heontugenyuf dopaminorgicneurunsIn A. Bartke & L.L. Murphy (Eds.), Neurobiology andneurophys¿ologyof cannahinoids:Vol IV Biochemistryandphysiologyof substanceabuse (pp. ¡19-164). BocaRalon,Florida, USA: CRC Press.

Ende.E..Barg,1., Levy, R.. Saya,O.,Hetdman.1., MeehouLam,R.,& Vogel, Z. (¡995). Low dusesof anandamidesinhibit

pharmaeulogicalefrecísof A9-tetrahydro-eannabinol.JournalofFharmacologyand ExperimentalTherapeutics,272, 699-707.

Ende,E.. & Mechoulam,R. (1993). Pharmaculogicalactivity ofthe cannabinoidreceptor agonisí anandarnide,a brainconstituen[. EuropeanJoanialof Pharmacology231, 313-314.

Ende, E., & Mechuulam,R.(1996a).Develupmenlalaspecísufanandamide:ontogenyof responseand prenatalexposure.Psychuneuroendocrinology21, 157-172.

Ende,E., & Mechoulam.R.( 1996b).Oníogene[ic developmentufthe responsetu anandamideand &-totrahydrocannabinolinmice. DevelopmeníalBrain Research, 95, ¡31-134.

Friod, PA. (1995).The Ottawaprenatalprospectivostudy(OPPS):melbudutogicalissuesandfindings. [lis easytu ttxrow [hebaby out with [hebath waletLife Selences.56. 2159-2168.

Gagin,R., Kouk, N., Cohen,E., & Shavit.Y (¡997). Prenatalmorphineenhancesmurphine-conditionedplacepreferencein adult rats.PhannacologyBioche,nistryant! Behavior58, 525-528.

Gaoni,Y.. & Mechou¡am,R. (1964). Isolation, structureelucidation,and partial synthesisof an activeconstitueníof hashish.JournalofAmericanChemicalSociety,86. ¡ 646-1647.

García,L., de Miguel, R., Ramos,JA., & Fernández-Ruiz,J.J.(1996). Perinatal-delta-9-letrahydrucannabinolexpusurein ratsmodifiesIhe responsivenessof midbrain dopaminergieneurunsin adulíhuodtu a variely of challonigeswith dupaminergie

drugs.Dm8andAlcohol Dependence,42, 155-166.

Oardner,EL., & Luwinson, J.H. (1991), Marijuana’s interactionwith brain reward systemsupdate 1991. PharmacologyBiochemistryandBehavion40, 571-580.

Oueudet,C., Santucci ,V., Rinaldi-Carmona,M.. Suubnié, P., & Le

Fur, 0. (¡995).‘[he GB1 cannabinuidreceptoraníagunistSR141716AaffectsA9 dupamineneuronalactivity in Ihe rat.NeuroReport, 6, 1293-1297.

Ha¡ikas,JA., We¡ler, RA.. Muuse,CL., & Hoffman, RA. (1985).

A longitudinal studyof marijuanacifecís.internationalJournnlof Addiction, 20, 701-711.

Hainmer,R.P. Jr (1985).‘[he sox-hormone-dependentdevelopment

of opiatereceptuisin the ral medial preuptic area. BrainResearch,360, 65-72,

Island, Til., Stinus, L., & Le Mual. M. (1989). Differential

mechanismsin theacquisitionandexpressiunuf horoin-inducedplacepreference.Psychopharmacology, 98, 61-67.

Harfslrand.A., Fuxe,K.. Cintra,A., Agnati, LE, Zini, 1., Wilkslrom,A.G., Okret, 5., Zhao-Ying,Y, Ouldstein,M.. Steinbusch,H.Venhustad,A., & Gustaffson,JA. (¡986). Glucocurticoidreceptorimmunureactivity in monoaminergieneuronsin rat brain.FroceedingsNoiiono! AcudemyofSciencesUSA, 83, 9779-9783.

55

NAVARRO AND RODRÍGUEZDE FONSECA

Herkenham,M., Lynn. AB., Little, MD., Johnson,MR.. Melvin,

LS., De Costa,BR., & Rice, K.C. (1990). Cannabinuidreceptorlocalizationin brain. ProceedingsNationalAcademyof SciencesU.S.A.,87, 1932-1936.

Holson, R.R., & Pearce,E. (1992). PrincipIesandpiífa¡ls in Iheanalysisof prenataltreatmenleffectsin mu¡[iparous species.Neurotoxicologyant! Teratology14, 221-228.

Hulchings,DE., Martin. BR., Gamagaris,Z., Miller, N., & Pico,‘[(¡989). Plasmaconcontrationsuf dolla-9-telrahydrocannahino¡

in damsandfetusesfullowing aculeur multiple prenataldusingin rals. Life Sciences,44, 697-701.

Hutchings,DE., Morgan,B., Hrake, SC., Shi, T., & Lasalle, E.(1987).Delta-9-tetrahydrucannabinulduring prognancyin Iheral: 1. Differential elfecísun maternalnuldtjon, ombiyotoxicity,

andgruwth in Ihe uffspring. Neurotoxicologyant! Teratology.9, 39-43.

Insel, T.R., Kinsloy, CH.. Mann. PE., & Bridges, RS. (1990).Prenatalstresshaslong- termcifecís un brain opiaterecepturs.Brain Research,511, 93-97.

Jakubovic,A.. Hattori, ‘[., & Mc Geer,PL. (¡977). Radioactivityin suckíedratsafler giving 4C~lelrahydrucannabinoltu Ihemuther.FuropeanJournal of Pharmacology,22, 221-223.

Keller, R.W. ir., Lefevre, R., Raucel,J.. Carlson,J.N., & Glick,S.D. (1996). Enhancedcucaineself-administratiunin adulí ratsprenalallyexposedtu cucaino.NeuroscienceLeíters, 205, ¡53-

156.Keshet,0.1., & Weinstuck,M. (1995). Maternalnaltrexoneprevenís

morphologicalandbehavioralalteraíiunsinduced in ratsbyprenatalstress.FhannacologyBiochemistryant! Behavio~50,413-419.

Koub, OF.. & Le Moal, M. (¡997). Drug abuse: hedunichumeostatiedysregulation.Science,278, 52-58.

Limar, AM., Haney, M., Becker, 1., Thompsun,ML., Kream,R.M., & Miczek. K. (1990).Fifecíuf earlyexposuretu delta-9-totrahydrocannabinolun ihe levols of opioid peptides,gunadolxuphin-releasinghormone,and substancoP in Ihe adultmalo rat brain. Brain Research,525, 78-83.

Lee, 5., & Rivier, C. (1996). Genderdifferencesin [heeffecí ufprenatalalcoholexposureun [hehypulhalamic-pituitary-adrenal

axis responselo inimune signals.Psychoneuroendocrinology,21, ¡45-155.

Macean,5., Piazza,PV., Deminiere,J.M., Lemaire,V., Monede,E, Simon, H.,Angelucci. L., & Le Mual M (¡991).Life events-induceddecreaseof curticosteroidtype-I receptursis assuciatedwith reducedcorticosíeronefeedbackaidenhancedvulnerabititytu amphetamineself-adminislration.Brain Research,547, 7-12.

Martin, 5., Crespo.JA., Ferradu,R., Oarcía-Lecumberri,C., Oil,L.. Ramos,JA.. Fernández-Ruiz.J.J.,Diez, N.. Manzanares.J.. & Ambrosio, E. (1996). Effects of Della-9-THCperinataltreatmentof mulhersun morphineaud fuodoperaníreinforcedbehaviorsin [he adulí offspning. Socieryfor Neuroscience

Abstract,22. 167.Martín-Calderón,J.L.,Muñoz, R.M., Vitíanda, MA., delArco, 1.,

Moreno,J.L.. RodríguezdeFonseca,E. & Navarro,M. (¡998).

Characíerizationof Iho acuteendocrinoacliunsof ¡-IU-2 ¡0, aputoní synthetic cannabinoidin rats. EuropeanJournal ufFharmacology(in pross).

Matsuda,LA., Lulail, S.J., Brownstein,Mi., Yuung, AL., &Bunner, TI. (1990). Síructurouf a cannabinuidreceptorandfuncíjonalexpressionof Ihe clonodcDNA. Nature, 346, 561-

564.

McCormick, CM., Smytho,J.W., Sharma,5., & Moaney, M.(1995). Sex-spociticeffectsof prenatalstressun hyputhalamic-pituitary-adrena¡responsostu slressand brain glucocurticoidreceptordensity in adult raís. Brain Research, 84, 55-61.

McEwen,B.S. (1987).Steruid hormonesandbrain deve¡opment:sumeguidelinesfor understandingactiunsuf pseudohormonosami olber toxio agonrs.Environmentalami HealthPerspectives,74, 177-192.

Mochoulam,R. (¡986). The pharmacohistoryof Cannabissativa.In R. Mechoulam(Ed.),Cannahinoidses TherapeuticAgents

(PP. ¡-19). Buca-Raton,Florida, USA. CRC Press.Mechoulam, R., Ben-Shabaí,5., Hanus, L., Ligumsky, M.,

Kaminski,NE., Schatz,AR., Gopher,A., A¡mog. 5., Martin,BR,, Compton,DR., Pertwoe,RO., Oriffin, O.. Bayewitch,M., Barg. J., & Vogol, Z. (1995). Identification of anondogenous2-monuglyceride.preseníin caninogut, thaI bindstu cannabinoidreceptors.Bioche,nicalPharrnacology,50. 83-90.

Mechoulam,R., Landor, N., Srebnik,M., Breuer,A., Sogal,M..Feigenbaum,Ji., Jarbo,T.U.C., & Consroe, E (¡987).Stereochemicalrequiremenísfor cannabimumetieaclivity. InRS. Rapaka& A. Makriyannis (Eds.),Structure-acrivityrelationshipsof tIte cannabinoids(PP. 15-30).Rockville, MD,

USA. NIDA rosearchmonograph79.Mirmiran, M., & Swaab,D.F. (¡987). Influenceof drugsun brain

nourotransrnissiunand behavioralsíagesduring developmont.Deve/opmenzof FharmacologvTherapeutics,10, 377-384.

Molina, VA., Wagner,i.M., & Spear,LP (1994). ‘[he behaviuralresponselo stressis alteredin adulí rats exposedperinataulytu cocaine.Fhysiologyant! Behavior, 55, 94¡-945.

Molina-Holgado,E, Molina-Holgado,E., Loreí,ML., CIonzáloz,MI., & Roader,TA. (¡993). Distribution uf indoleaminosaud

L3HI-paruxetinebinding in ral brain regionfollowing acutoorperinatalde¡ta-9-totrahydrocannabinoltroatment.s.Neurochenticol

Research,18, 1183-1191.Navarro,M., Fernández-Ruiz,J.J.,do Miguel, R., Hernández,ML.,

Cohoira, M., & Ramos,JA. (1993a).An aculedoso uf ñ~-leírahydrocannabinolaffocts bohaviuraland neuruchemical,ndicesof mesolimhicdopaminergieactivity. BehaviouralBrain

Research,5, 37-46.Navarro,M., Fernández-Ruiz,Ji., deMiguel. R., Hernández,ML.,

Cobeira,M., & Ramos,JA. (1993b). in Motor disturbancesinducedby an aculedoseof A9-tetrahydro-cannabino¡:possibleinvolvemení of nigrosírialal dopaminergie allerations.Fhar~nacologyBiochernistryant! BehaVior,45, 291-298.

Navarro,M., Hernández,E., Muñoz, R.M., delArco, 1., Villanda,MA., Carrera,M.R.A,, & Rodríguezde Fonseca,E (1997).Acule administration of the GB-1 cannabinoidreceptor

56

CANNAHIS AND ADDICTION VULNERABtLITY 57

antagunisí5R141716Ainducesanxiely-like responsesin [herat. NeuroReporr,8,491-496.

Navarro, M., Martín-Calderón,J.L., delArco, 1., Villanda, MA..Sánchez,L, Muñoz, R.M., Orengo,E., Gapdevila,E., Ghuwon,J., & Rodríguezde Fonseca,E (in press).Fifocís of subchronicIrealmeníwith dupaminergicagonisísandantagonisíson Ihe

acule sensitivity tu cannabinoid expusure: behavioral.neurochemicalandhormonalsludies. In T. Archer,R. Boninger,

&T. Palomo(Eds.),Strareg¡esfor StudyingBrain Disorders,Vol. IV. Madrid: Cerebroy Monte.

Navarro,M., de Miguel, R., Rodríguezde Fonseca,E?, Ramos,JA.,

& Fernández-Ruiz,J.J. (1996). PerinatalcannabinoidexposuremodifiesIhe sociusexualappruachbohaviorandIho mesolimbicdopaminorgicacíiviíy of adulí malo rats. BehaviouralBrainResearch,75, 9 1-98,

Navarro,M., Rodríguezdo Fonseca,E, Hernández,ML., Ramos,JA.. & Fernández-Ruiz,Ji. (1994a). Changosin Ihe adultmotor bebaviorlblluwing perinatalcannabinoidexposureliirats: involvemont uf nigrosiriatal dupaminergicactivity.

PharmacologsBiochemistryant! Behavior,47, 47-58.Navarro, M., Rubio, P,, & Rodríguezde Fonseca,E. (1994h).Sox-

dimorphic psychumotoraclivalionaflor perinataloxposurelo(-)-A9-totrahydrocannabinul.An ontogeniestudyin wistar rats.Psychopharmacuiogy,116, 414-422.

Navarro, M., Rubio, P., & Rodríguezde Fonseca,F. (¡995).

Hohaviuuralconsoquencesof maternalexposurotu cannabinoidsin rats.Psychopharrnacology,/22, ¡-14.

Piazza,PV., Deminiero,SM., Le Mual, M., & Simon, Fi. (1989).FaclursthaI predicí individual vulnerability tu amphetamineself-adn,inistration.Science,245, l5ll-1513.

Piazza,PV., ¡Joroche,y., Deminiéro,i.M., Maccari,5., Le Moal,

M., & Simon, Fi. (1993).Corticosteronoin Iho rangouf stross-induced¡evels pussossesroinforcing propertios:Implicationsfor sonsatiun-seekingbehaviors.ProceedingsNationalAcademyof SciencesUSA, 90, 11738-11742.

Piazza,PV., & Le Moal, M. (1996). Palhophysiologicalbasis ofvt¡lnerability Lo drug abuso: role of un interactionborweonsíress,glucucurticoids,anddupaminergieneurons.AnnualReviewof Pharmacologyant! Toxicology,36, 359-378

Potíyrov, T., & Weinstock,M. (¡997). Effect of prenatalstressun opiuid componeníof exploralionin differeníexperimentalsítuations.Phar,nacologyBiochemistryant! Behavior,58,

387-393.Rinatdi-Carrnona,M., Barth, E, Hoaulme,M., Shiro, D., Calandra,

B., Congy,C., Martines, 5., Maruani, J., Nelial, CI., Capul,D.,Ferrara,P., Soubrie, E. Breliore, J.C., & Le Fur,0. (¡994).SR 141716A, a potentand selectivoanlagonisíof Ihe braincannabinoidreceptor.FEBS¡caen,350, 240-244.

Robins,L.N., & Milis, iL. (¡993). Effects uf in ulero exposurelo streetdrugs.AmericanJournal of Fublíc fleo/fi,, supplement

tu Vol 83, 8-32.Rodríguezdo Fonseca,F., Carrera,M.R.A., Navarro, M., Koob,

OF., & Weiss, F. (1997).Activalion of corticoíropin-releasingfactor in Iho limbie systemduring cannabinuidwithdrawa¡.

Science,276, 2050-2054.

Rodríguezde Fonseca,E., Cebeira,M., Fernández-Ruiz,i.J.,

Navarro, M.., & Ramos,JA. (1991a).Effocts uf pro- andperinataloxpusuretu hashishexlractsun theontogenyof braindopaminergicneuroas.Neuruscience,43, 713-723.

RodríguezdeFonseca,E, Ceboira,M., Hernández,ML., Ramos,JA., & Fernández-Ruiz,ii. (¡990). Changes in brain

dopaminorgic indices indueed by perinalal exposuro locannabinuidsin rats. DevelopmentalBrain Research.51, 237-24

Rodríguezde Fonseca,EA., Ceheira,M., Ramos,JA., Martín,M., & Fernández-Ruiz,J.J. (1994b).Cannabinoidreceptorsinral brainarcas:sexualdifferences,fluctuationsduring ostruus

cycte, and changos after gonadectumyand sez steruidreplacement.Life Sciences,54,159-170.

Rodríguezde Fonseca,E., Fernández-Ruiz,Ji., Eldridge, J.C.,Síeger,R.W., Eartke,A., & Murphy, L.L. (1991b).EffeclsofIho oxpusuretu de¡la-9-íeírahydru-eannabinolun Ihe adronalmedultaryfunetion:ovidonceof an aculecifecíauddevetopmont

uf tolerancein chronictreatmonts. Pharmaco/agy Biochemistry

ant! Behavior, 40, 593-598.Rodríguezde Fonseca,E, Fernández-Ruiz,Ji., Murphy, L.L..

Ceboira,M., Steger,R.W., Bartke,A., & Ramos,JA. (1992b).Acule offecls uf A9-reírahydrocannabinotun dopaminergicacíivity in severa¡ral brain area.s.FharmacologyBiochemistry

ant! Behavior, 42, 269-275.Rodríguezde Fonseca,E, Hernández,ML., do Miguel, R..

Fernández-Ruiz,i.J., & Ramos,JA. (1992a).Early changosin Ihe developmontof dopaminergieneurotransmisgionaftormaternalexposuretu eannabinoids.PharniacologyBiochemísrryant! Behavior, 41, 469-474.

Rodríguezde Fonseca,E, Martín-Calderón,iL., Mechoulam,R.,& Navarro,M. (¡994a).Ropeatedstimulalion of DI dopamine

rocoptorsenhances(-)-l 1 -hydroxy-A5-letrahydrocannabinol-dimethythepíyl-inducedcatalopsyin malo rats.NeuroReport,5,761-765.

RodríguezdeFonseca,E, Ramos,JA., Bonnin,A., & Fernández-Ruiz, Ji. (1993). Presonceof cannabinoidbinding sitesin Ihe

brain from earlyposinatalagos.NeumReport,4, 135-138.RodríguezdeFonseca,E., Rubio, E. Martín-Calderón,J.L.,Came,

S.B., Koob. OF., & Navarro.M. (1995a). Tho dopaminereceptoragonisí7-OFl-DPAT modulatesIbe acquisitionandoxprossionof morphine-inducedplacepreference.FuropeanJoarnal of Pharrnacoiogy,274,47-55.

RodríguezdeFonseca,E, Rubio, P.. Menzaghi,E, Mer¡u-Pich,E.,Rivior, J., Koob, OF?, & Navarro,M. (1996). Curticotropin-reloasingfactor (CRF) aníagunisí ID- Phel2, Nle2l,38,

CMeLeu37]CRF atíenuatostho aculeacíionsuf the highlypotenícannabinoidreceptoragonist HIJ-210 un defensivo-withdrawal bohaviorin rats. Journalof Pharmacologyant!

ExperimentalTherapeutics,276, 56-64.Rodríguezde Fonseca,F.. Villanda, MA.. Muñoz, R.M., San-

Martin-Clarke,O..& Navarro,M. (1995b).Diríerontialeffectsof chronic lreatmontwith eiíherdopamineD ¡ ur D2 receptoragonisísun Iho aculeneuroendocrinoaclionsuf the highly

potoní synthetic cannabinuid HU-210 in malo rats.Neuroent!ocrinology,61, 714-721

NAVARRO AND RODRÍGUEZDE FONSECA

Romero,J., García-Palomero,E., Lin, 5.. Ramos.JA., Makriynnis,A., & Fernández-Ruiz,Ji. (1996). Exírapyramidaloffecls ofmothanandamide,ananalogueuf anandamido,Ihe endogenuusCBI receptorligand. Life Sciences,.58, 1249-1257.

Rosenkrantz,H., Spraguo,RA., Fleischman,R.W., & Braude,MC. (1975). Oral A9-totrahydrocannabinoltoxicity in rats

treatedfur periodsup tu siz months.Toxicologyant! Appliet!Pharmacology,32, 399-417.

Rubio, 1’., RodríguezdeFonseca,E, Martin- Calderón,J.L. del Arco,1., Bartolomé,5., Villanda, MA., & Navarro,M. (In press).Maternalexposurelo ¡uw dosesof A9-teírahydrocannabinolfacilitatesmorphine-inducedplacecondiíioningin adulí malooffspring. FharnwcologyBiochemisu-yant! Behavior.

Rubio, P, Rodríguezde Fonseca,F., Muñoz,R.M., Ariznavarreta,C., Martín- Calderón,iL., & Navarro,M. (1995). Long-termbehavioraleffect.sof perinalal exposuretu A9-tetrahydrocannabinolin rats: possibleroleuf piluiíazy-adrenalaxis. L~fe Sciences,56.

2 169-2176.Shaham,Y., & Stewart,J. (¡995). Stressreinstalesheroin-seeking

in drug-free animals: an effect mimicking heroin, not

withdrawal.Psychophartnacology,119, 334-341.Síella, N., Schweitzer, P., & Piomolil, D. (1997). A secund

endogenouscannahinoidthat modulaleslong-termpotentiation.

Nature, 388, 773-778.Taylor, AN., Branch,B.J.,Nelson,L.R., Fane,LA., & Po¡and,

RE. (¡986). Prenatalcíhanoland ontogenyof pituitary-adrenalresponseslo ethanolaidmorphine.Alcohol, 3, 255-

259.

Valleé, M.. Mayo, W., De¡lu, E, Le Moal, M., Simon, Fi., &Macean (¡997). Prenatalstressinduceshigh anxiety andposínatal hand¡inginduceslow anxiety in adult offspring:correlationwith slress-inducedcorticusteronesecretion.TIte

icurnal of Neuroscience,¡7, 2626-2636.Vela, O., Fuentes,JA., Bonnin, A., Fernandez-Ruiz,J., & Ruiz-

Gayo, M. (¡995).Perinatalexpusuretu A9-telrahydrocannabinol

leadslo changosin opioid-relatedbehavioralpaíternsin raís.Brain Research,680, 142-147.

Wallers, D.F., & Can,LA. (¡986). Changosin brain calecholaminemechanismsfollowing perinatal oxposuretu marihuana.FharínacologyBiochemisrryant! Behavior, 25, 763-778.

Ward, IL., & Weisz.J. (¡984).Differenlial effectsuf maternalstressun circulating levelsof curlicusterono,progesteroneandtestosteronein malo andfemaloral fetusesandtheir mothers.Ent!ocrinology, 114. 1635-1644.

Wenger,T., Fragkakis,O., Oiannikou,E?, & Yiannakis,N. (1997).The effoctsuf prenatallycidministeredendogenouscannabinoidon rat offspring. PharmacologyBiochemistryant! Behavior,58, 537-544.

Zuckorman,B. (1991). Drug effects- A searchfor mechanisms.In M.N, Kilbey andK. Asghar(Eds.),MothudologicalissuesIn controlledstudiesun effectsof prenatalexposurelo drugabuse(PP 352-362).NiDA ResearchMonograph 114.

ReceivedOctubor6, ¡997

Revision receivedFebruary12, 1998AcceptedMarch 9, 1998

58