Influence of the anabolic-androgenic steroid nandrolone on cannabinoid dependence

19 Pages·00.45 MB·English

Neuropharmacology50(2006)788e806
www.elsevier.com/locate/neuropharm
Influence of the anabolic-androgenic steroid nandrolone
on cannabinoid dependence
Evelyne Ce´le´riera, Therese Ahdepil b, Helena Wikanderb, Fernando Berrendero a,
Fred Nybergb, Rafael Maldonado a,*
aLaboratoriofNeurofarmacologia,FacultatdeCie´nciesdelaSalutidelaVida,UniversitatPompeuFabra,
C/DoctorAiguader80,08003Barcelona,Spain
bDepartmentofPharmaceuticalBiosciences,UniversityofUppsala,Box591Biomedicum,S75124Uppsala,Sweden
Received8April2005;receivedinrevisedform29November2005;accepted29November2005
Abstract
Theidentificationofthepossiblefactorsthatmightenhancetheriskofdevelopingdrugaddictionandrelatedmotivationaldisordersiscrucial
to reduce the prevalence of these problems. Here, we examined in micewhether the exposure to the anabolic-androgenic steroid nandrolone
wouldaffect thepharmacological and motivational effects inducedby D9-tetrahydrocannabinol (THC), the principalpsychoactive component
of Cannabis sativa. Mice received nandrolone using pre-exposure (during 14days before THC treatment) or co-administration (1h before
eachTHCinjection)procedures.Bothnandrolonetreatmentsdidnotmodifytheacuteantinociceptive,hypothermicandhypolocomotoreffects
of THC or the developmentof tolerance after chronic THC administration. Nandrolone pre-exposure blocked THC- and food-induced condi-
tionedplacepreferenceandincreasedthesomaticmanifestationsofTHCwithdrawalprecipitatedbytheCB1cannabinoidantagonistrimona-
bant (SR141617A). The aversive effects of THC were not changed by nandrolone. Furthermore, nandrolone pre-exposure attenuated the
anxiolytic-likeeffectsofalowdoseofTHCwithoutalteringtheanxiogenic-likeeffectsofahighdoseinthelit/darkbox,openfieldandelevated
plus-maze.BiochemicalexperimentsshowedthatchronicnandrolonetreatmentdidnotmodifyCB1receptorbindingandGTP-bindingprotein
activationinthecaudate-putamenandcerebellum.Takentogether,ourresultssuggestthatchronicnandrolonetreatmentaltersbehaviouralre-
sponsesrelated to cannabinoidaddictive properties.
(cid:1)2005Elsevier Ltd.Allrightsreserved.
Keywords:Anabolic-androgenicsteroid;Nandrolone;THC;Dependence;Anxiety;Mice
1. Introduction (AAS),usedasdopingsubstances,asariskfactorforthecon-
sumption of drugs of abuse such as cannabis.
Considerable efforts are now devoted to the study of risk Derivates from Cannabis sativa, whose main psychoactive
factors that may increase individual vulnerability to drugs of constituent is the D9-tetrahydrocannabinol (THC) (Mechoulam
abuse. An important factor that could enhance the risk for etal.,1970),are today the most consumed illicit drugs world-
an addictive process and motivational related disorders is the wide (Smart and Ogborne, 2000). Cannabinoid effects are
exposure to pharmacological compounds able to modify the mediatedbytheactivationoftworeceptors,theCB1cannabi-
physiological equilibrium of the rewarding system (Koob noid receptor highly abundant in the central nervous system,
and Le Moal, 2001). The purpose of the present study was and the CB2 receptor, mainly located in the cells of the
to investigate the effects of anabolic-androgenic steroids immune system (Ameri, 1999). The cannabinoid system is
closely related to several neurobiological pathways involved
in motivation, mood and addictive behaviours, particularly
the dopaminergic and the opioid system (for reviews, see
Manzanares et al., 1999; Maldonado and Rodr´ıguez de
* Correspondingauthor.Tel.:þ34935422845;fax:þ34935422802.
E-mailaddress:[email protected](R.Maldonado). Fonseca). Accordingly, cannabinoids have been reported to
0028-3908/$-seefrontmatter(cid:1)2005ElsevierLtd.Allrightsreserved.
doi:10.1016/j.neuropharm.2005.11.017
E.Ce´le´rieretal./Neuropharmacology50(2006)788e806 789
produce behavioural and biochemical changes in animals treatmentwithTHCwereevaluated.Inordertomimichuman
similar to other drugs of abuse and leading to dependence abuseregimes,supraphysiologicdosesofnandroloneweread-
(Maldonado and Rodr´ıguez de Fonseca, 2002). However, ministeredusingtwodifferentprotocols,i.e.co-administration
one important aspect of marijuana activity is the complexity during THC treatment and pre-exposure before THC
of evoked emotional/affective responses and the possibility treatment.
of dual euphoric-dysphoric effects (Halikas et al., 1985). In-
deed,reinforcingandaversiveaswellasanxiolyticandanxio- 2.Methods
genic effects have been reported after the administration of
THC and other cannabinoid agonists (Chaperon and Thie´bot, 2.1.Animals
1999; Ghozland et al., 2002; Berrendero and Maldonado,
2002). Albino male CD-1 mice (CRIFFA, France) weighing 20e22g were
housed five per cages and maintained at a controlled temperature
AAS are synthetic derivates of testosterone widely used in
(21(cid:1)1(cid:2)C)andhumidity(55(cid:1)10%)environment.Themiceweregivenac-
theclinicasandrogen replacementtherapy andaschemother-
cesstofoodandwateradlibitum.Lightingwasmaintainedat12-hcycles(on
apy for certain types of cancer (Wilson and Griffin, 1980). at7a.m.andoffat7p.m.).Alltheexperimentswereperformedduringthe
During the last five decades, AAS has been used at doses lightphaseofthedark/lightcycle.Theanimalswerehabituatedtotheexper-
10e100 times the therapeutic range by many athletes and imentalroomandhandledfor1weekbeforethestartoftheexperiments.All
animalproceduresmettheguidelinesoftheNationalInstituteofHealthde-
bodybuilders to enhance their physical performance, increase
tailedinthe‘‘GuidefortheCareanduseofLaboratoryAnimals’’,theEuro-
muscle mass and intensify training regimens (Wilson, 1988;
peanCommunitiesdirective86/609/EECregulatinganimalresearchandwere
Lukas, 1993; Yesalis and Bahrke, 1995). The chronic use of approvedbytheLocalEthicalCommittees.Allexperimentswereperformed
highdosesofAAShasbeenreportedtocauseseveralphysical withtheinvestigatorsbeingblindtothetreatmentconditions.
andpsychologicalside-effectssuchasliverdysfunction,coro-
naryheartdisease,reproductivedysfunction,acne,depression, 2.2.Drugs
personality changes and aggressive behaviour (Williamson
and Young, 1992; Pope and Katz, 1994; Bahrke et al., THC and the AAS nandrolone decanoate were purchased from Sigma
(Poole, UK). The selective CB1 cannabinoid receptor antagonist
1996). Interestingly, a concurrent abuse of AAS has also
SR141617A (rimonabant) was generously provided by Sanofi Research
been reported among addicts and others not connected to
(France).Nandrolonewasdissolvedinvehicle(10%ethanol/10%cremophor
sports (DuRant etal.,1993;Lukas, 1993;Yesalis andBahrke, EL/80% distilled water) and injected intramuscularly (i.m.) in a volume of
1995; Kindlundh et al., 1999). Several studies have suggested 2ml/gbodyweight.THCwasdissolvedinvehicle(5%ethanol/5%cremophor
theassociationbetweenuseofAASandconsumptionofalco- EL/90% distilled water) and injected intraperitoneally (i.p.) in a volume of
10ml/g body weight. Rimonabant was dissolved in vehicle (10% ethanol/
hol, tobacco and illicit drugs, such as cannabis, opiates, am-
10% cremophor EL/80% distilled water) and injected i.p. in a volume of
phetamine and ecstasy (DuRant et al., 1993; Yesalis and
20ml/gbodyweight.Controlmicereceivedequivolumicvehicleinjections.
Bahrke, 1995; Kindlundh et al., 1999; Kanayama et al.,
2003). Based on these clinical and epidemiological studies,
2.3.Behaviouralexperiments
AAS exposure has been proposed to serve as a ‘‘gateway’’
for the misuse of other drugs of abuse (Arvary and Pope, The effects of nandrolone treatment on acute and chronic THC effects
2000). In line with this hypothesis, recent animal studies were evaluated. Two different protocols (pre-exposure or co-administration)
have shown that AAS can be self-administered by laboratory were used for nandrolone administration in an attempt to mimic conditions
similartothoseusedduringhumanabuseregimes:drugintakeconcomitant
animals (Ballard and Wood, 2005). AAS have also been re-
tonandroloneuseordrugintakewithapastoflong-termconsumptionofnan-
ported to evoke neurobiochemical and behavioural alterations
drolone.Inthefirstprotocol(pre-exposure),nandrolonewaschronicallyad-
related to dependence, mood and motivation in rodents espe- ministered once daily during 14days before starting THC treatment. In the
cially by affecting the endogenous opioid and dopamine sys- secondprotocol(co-administration),nandrolonewasadministered1hbefore
tem (Lukas, 1993; Menard et al., 1995; Clark et al., 1996; eachTHCinjection.Thei.m.injectionsofnandroloneweregivenalternatively
intheleftandtherighthindlegwhenrepeatedadministrationwasrequired.A
Le Greve`s et al., 1997; Johansson et al., 1997, 2000a,b;
supra-therapeutic dose of nandrolone (15mg/kg) was chosen (i) because it
Thiblin et al, 1999; Hallberg et al., 2000; Schlussman et al.,
mimicsthedoseself-administeredbyheavynandroloneabusers(Williamson
2000; Ce´le´rier et al., 2003; Kindlundh et al., 2003 and for re- andYoung,1992)and(ii)becauseithasbeenpreviouslyshowntoinducebio-
view, see Clark and Henderson, 2003). chemicalchangesintheendogenousopioidanddopaminesysteminrodents
Interactions between sex-steroids and the endocannabinoid (Johanssonetal.,1997).
system on reproduction and endocrine responses have been
well documented (Gonzalez et al., 2000; MacCarrone et al., 2.3.1.AcuteeffectsofTHC
TheacuteeffectsinducedbydifferentdosesofTHC(5,10or20mg/kg,
2000; Corchero et al., 2001). However, the possible influence
i.p.) on nociception, locomotion, and rectal temperature were evaluated in
oftheseinteractionsonthebehaviouralresponsesmediatedby
eachexperimentalgroup.
the central nervous system remains to be elucidated. In the Twonociceptivetestswereused,thetail-immersionandthehot-plate.In
present study, we use different behavioural models in mice thetail-immersiontest,miceweregentlyplacedinarestrainercylinder.The
to evaluate the effects of the AAS nandrolone on several nociceptive threshold was assessed as described previously (Janssen et al.,
1963),bymeasuringthetimetowithdrawthetailimmersedinathermostated
THC behavioural responses related to its addictiveproperties.
waterbath(50(cid:1)0.1(cid:2)C)(Clifton-ScientificInstruments,England),withacut
Forthispurpose,theeffectsofnandroloneexposureonthebe-
offlatencyof15stopreventtissuedamage.Nociceptiveresponseswerealso
haviouralandsomaticresponsesinducedbyacuteandchronic measuredusingahot-plateanalgesiameter(Colombus,Ohio,USA).Aglass
790 E.Ce´le´rieretal./Neuropharmacology50(2006)788e806
cylinder(19cmhigh,19cmdiameter)wasusedtokeepthemiceontheheat- 523s; mean time spent in dotted compartment: 553s). After this session,
edsurfaceoftheplate,whichwasmaintainedatatemperatureof52(cid:1)0.1(cid:2)C. micewererandomized for pairingto drug orvehicle administration andfor
Thelatencyforjumpingwasevaluatedasthenociceptivethreshold,withacut- assignment to a compartment. Care was taken to ensure that treatments
offtimeof240s. werecounterbalancedascloselyaspossiblebetweencompartmentstoensure
Locomotor responses were evaluated using locomotor activity boxes anunbiasedprocedure.ToevaluateTHCrewardingeffects,micewereexposed
(9(cid:3)20(cid:3)11cm; Imetronic, Lyon, France). The boxes contained a line of toapriminginjectionofTHC(1mg/kg)24hbeforestartingtheconditioning
photocells2cmabovethefloortomeasurehorizontalmovementsandanother phase,aspreviouslyreported(ValjentandMaldonado,2000).Duringthecon-
linelocated6cmabovethefloortomeasureverticalactivity(rearing).Onthe ditioningphase,micewerealternativelytreatedfor10consecutivedayswith
experimentalday,themicewereindividuallyplacedintheboxesandtheam- THC(1mg/kg)orvehicle.ToevaluateTHCaversiveeffects,micewerenot
bulatory,horizontal(ambulatorymovementsplussmallmovements)andver- exposedtoanypriminginjectionandwerealternativelytreatedfor10consec-
ticalactivitieswererecordedduring10mininalowluminosityenvironment utive days withTHC (5mg/kg) or vehicle during the conditioning phase as
(5e15lx). previouslyreported(ValjentandMaldonado,2000). Micein thedruggroup
Rectaltemperaturewasmeasuredineachmouseusinganelectronicther- receivedTHCondays1,3,5,7and9intheirassignedcompartment,andve-
mocoupleflexibleprobe(Panlab,Barcelona,Spain).Theprobewaslubricated hicleondays2,4,6,8and10intheoppositecompartment.Controlmicere-
andplaced3cmintotherectumofthemicefor20sbeforethetemperature ceivedvehicleinbothcompartmentsalternatively.Doorsmatchingthewalls
wasrecorded. ofthecompartmentallowedtheconfinementofthemicefor45minimmedi-
Inordertohabituatetheanimalstothetestenvironmentandtoobtainasta- atelyafterTHCorvehicleinjection.Finally,thetestphasewasconducted24h
blebaseline,tail-immersionresponsewasmeasuredfor2daysbeforetheex- afterthelastconditioningsessionexactlyas wasthepreconditioningphase,
periment. On the experimental day, basal tail-immersion latency and rectal i.e.withfreeaccesstobothcompartmentsfor20min.
temperaturewereevaluatedbeforestartingthetreatment.Locomotoractivity Foodrewardwasevaluatedinanimalswithlimitedaccesstofoodandha-
wasevaluated20minafterTHCinjectionduring10min.Thetail-immersion bituatedtosucroseconsumptionfor5daysbeforethetest.Afixedamountof
testwasperformed30minafterTHCinjectionandwasimmediatelyfollowed foodequivalentto15%ofmousebodyweightwasappliedperday,asprevi-
by the hot-plate test. Rectal temperature was measured 45min after THC ouslyreported(Elmeretal.,1995).Animalsweremaintainedonthesamediet
injection. throughout theexperimentalperiod.Duringthepreconditioningphase,mice
wereplacedinthemiddleoftheneutralareaandtheirlocationwasrecorded
for18min.Miceconditionedtofoodhadfreeaccesstoit(normalmousefood
2.3.2.TolerancetoTHCeffects
plussucrose)intheconfinedcompartmentondays1,3and5,andhadnoac-
Asinthepreviousexperiment,thebasaltail-immersionresponsewaseval-
cesstofoodintheothercompartmentondays2,4and6.Controlanimalswere
uatedfor2daysbeforethebeginningofTHCinjections.THC(20mg/kg,i.p.)
alternativelyexposedtobothcompartmentsduringthisphaseandhadnoac-
wasadministeredtwiceaday(10a.m.and7p.m.)during5days.Everymorn-
cesstofoodintheassignedcompartment.Micewereconfinedinthecondi-
ingbeforeanyTHCinjection,bodyweight,tail-immersionlatencyandrectal
tioning compartments during a period of 20min. The testing phase was
temperature were measured. Locomotor activity was recorded 20min after
conducted24hafterthefinalconditioningsessionexactlyastheprecondition-
eachmorningTHCinjectionfor10min.Tail-immersionandrectaltempera-
ingphase,i.e.freeaccesstoeachcompartmentfor18min.
tureweremeasured30and45min,respectively,aftereachmorningTHCin-
InbothTHCandfoodexperiments,ascorewascalculatedforeachmouse
jection.Thehot-platetestwasonlyperformedthefirstandlastdayofTHC
asthedifferencebetweenthepost-conditioningandthepre-conditioningtime
treatment(day1and5),immediatelyafterthetail-immersionexposure.
spentinthedrug-pairedcompartment.
2.3.3.Rimonabant-precipitatedTHCwithdrawal
2.3.5.THCeffectsonanxiety-likeresponses
Mice chronically treated with THC (20mg/kg, i.p., twice daily during
The anxiolytic- and anxiogenic-like effects of THC were evaluated by
5days)fortheevaluationoftolerancetoitspharmacologicaleffectsreceived
using three experimental procedures: lit/dark box, open-field and elevated
anadditionalTHCmorninginjectiononday6(20mg/kg,i.p.).Fourhourslat-
plus-maze.Behaviouraltestswereperformed30minafterTHCadministration.
er,cannabinoidwithdrawalwasprecipitatedbyinjectionoftheselectiveCB1
Anxiolytic-likeeffectsofTHCwereinducedbyusingalowdose(0.2mg/kg),
receptorantagonistrimonabant(10mg/kg,i.p.).Themicewereplacedindivid-
whereasahigherdoseofTHC(7.5mg/kg)inducedtheanxiogenic-likeeffects.
uallyintotestchamberstoevaluatethebehaviouralsignsofwithdrawal.The
Inthelit/darkboxprocedure(Fillioletal.,2000),micewereindividually
chambersconsistedoftransparentroundplasticboxes(30cmindiameterand
exposed for 5min to a box consisting of a small dark compartment
50cminheight)withawhitefloor.Somaticsignsofwithdrawalwereevalu-
(15(cid:3)20(cid:3)25cm)withblackfloorandwalls,dimlylit(5e10lx)connected
ated15minbeforeand45minafterrimonabantchallenge.Thenumberofwet
bya4cmlongtunneltoalargerlitcompartment(30(cid:3)20(cid:3)25)withwhite
dogshakes,frontpawtremorsandsniffingwerecounted.Ptosiswasscored,1
floorandwalls,exposedtointenseillumination(500lx).Linesweredrawnon
forappearanceand0fornon-appearance,withineach5mintimeperiod.Tak-
thefloorofbothcompartmentstoallowmeasurementsoflocomotoractivity
ingintoaccountalltheindividualsigns,aglobalwithdrawalscorewascalcu-
bycountingthenumbersofsquares(5(cid:3)5cm)crossed.Theselinesalsodi-
lated for each mouse by giving each individual sign a relative weight as
videthelitcompartmentintothreeequalzones,fromthetunneltotheopposite
previouslyreported(Hutchesonetal.,1998).
wall,designatedasproximal,centralanddistalzones.Tostarttheexperiment,
eachmousewasplacedinthedarkcompartmentfacingthelitarea.Thepa-
2.3.4.RewardingeffectsofTHCandfood rametersrecordedwerelatencyforfirstentranceintothelitarea,timespent
TherewardingeffectsofTHCandfoodwereevaluatedbyusingthecon- ineachcompartment,numberofsquarescrossedineachcompartment,loco-
ditionedplacepreferenceparadigm.Theplacepreferenceapparatusconsisted motoractivity(numbertotalofsquarescrossed)andnumberofentriesineach
oftwodifferentcubiccompartments(15(cid:3)15(cid:3)15cm)separatedbyatrian- compartmentandintoeachzoneofthelitcompartment.
gularneutralarea(15cmperside).Thetwocompartmentshadvisualandtac- Theopen-fieldconsistedofarectangulararea(70cmwide,90cmlongand
tiledifferences,whereonecompartmenthaddottedwallsandroughfloorand 60cmhigh)brightlyilluminatedfromthetop(500lx).Atotalof63squares
theothercompartmenthadstripedwallsandsmoothfloor.Themovementand (10(cid:3)10cm)weredrawnwithblacklinesonthewhitefloordividingthefield
location of the mice were recorded by computerized monitoring software into central and peripheral areas. To start the experiment, each mouse was
(Smart(cid:2) Videotrack, Panlab., Barcelona, Spain) with images relayed from placedinthecentralareaofthefield.Thefollowingeventswererecordeddur-
a camera placed above the apparatus. The place preference conditioning inganobservationperiodof5min:latencyto gooutfromthe centralarea,
schedule consisted of three phases. During the preconditioning phase, the numbersofsquarescrossedineacharea,numberofrearing,numberofentries
mousewas placed in the middle of the neutral area and allowed to explore intothecentralareaandlocomotoractivity(totalnumberofsquarescrossed).
both compartments, and the time spent in each compartment was measured Theelevatedplus-mazeconsistedoftwoarms(length30cm;width7cm)
during20min.Noinitialpreferenceoraversionwasrevealedforanycompart- formingtheshapeofaplussituatedonaplatform80cmaboveground.Oneof
ment in any of the experiments (mean time spent in striped compartment: thearmswasopenandtheotherclosed(14cm).Numberofentriesandtime
E.Ce´le´rieretal./Neuropharmacology50(2006)788e806 791
spentintotheopenandclosedarmwererecordedduringanobservationperiod (nandrolone/vehicleandTHC/vehicletreatments).Whenrequired,thethree-
of5min. wayANOVAwasfollowedbycorrespondingtwo-wayandone-wayANOVAs.
Statistical analyses of data from CB1 receptor bindingand activation of
2.4.Biochemical experiments GTP-bindingproteinwereassessedbyusingtheunpairedtwo-tailedStudent’s
t-test.
StatisticalsignificancecriterionwasP<0.05inallcases.
CB1receptorbindingandactivationofGTP-bindingproteinwasinvesti-
gatedintwobrainareasrelatedtocannabinoidbehaviouralresponsesandde-
pendenceinanimals:thecaudate-putamenandthecerebellum(Navarroetal., 3. Results
2001;Castan˜e´ etal.,2004).Thestudieswereperformedinmicechronically
treated with nandrolone (15mg/kg) or vehicle once daily during 14days.
3.1. Effects of nandrolone exposure on acute THC
On day 15, mice were decapitated and their brains were quickly removed
andfrozenbyimmersionin2-methyl-butanesurroundedbydryice.Allsam- responses
ples were stored at (cid:4)80(cid:2)C during a similar short period of time until pro-
cessedforanalysis. Theinfluenceofnandroloneexposureontheacutepharma-
cological effects of THC(5, 10, and 20mg/kg,i.p.) was eval-
2.4.1.NandroloneeffectsoncannabinoidCB1receptor
uated by measuring the responses induced in nociception,
binding
body temperature and locomotor activity. Nandrolone
TheprotocolusedwasbasicallythemethoddescribedbyHerkenhametal.
(1991).Briefly,brainstructureswereincubatedfor2.5hat37(cid:2)Cinabuffer (15mg/kg, i.m.) or vehicle was administered using the two
containing50mMTriswith5%bovineserumalbumin(fattyacid-free),pH different protocols: co-administration and pre-exposure. Con-
7.4,and10nM[3H]CP-55,940(PerkinElmerLifeSciences)preparedinthe trol experiments showed that nandrolone had no intrinsic
same buffer, in the absence or presence of 10mM non-labelled CP-55,940
effect in any of the parameters studied when injected in
(Tocris) to determine total and non-specific binding,respectively. Following
combination with vehicle.
this incubation, slides were washed in 50mM Tris buffer with 1% bovine
serumalbumin(fattyacid-free),pH7.4,for4h(2(cid:3)2h)at0(cid:2)C,dippedin
ice-colddistilledwater,thendriedunderastreamofcooldriedair.Autoradio-
3.1.1. THC antinociceptive effects
gramsweregeneratedbyapposingthelabelledtissues,togetherwithautora-
Chronic nandrolone pre-exposure for 14days did not sig-
diographic standards ([3H]Microscales, Amersham), to tritium-sensitive film
(Hyperfilm-[3H], Amersham) for a period of 10days and developed for nificantly modify THC acute antinociceptive effects in the
4minat20(cid:2)C.Developedfilmswereanalysedandquantifiedinacomputer- tail-immersion and the hot-plate test (Table 1). In the tail-
izedimageanalysissystem(MCID,St.Catharines,Ontario,Canada)usingthe immersion test, significant antinociceptive effects of 10and
standardcurvegeneratedfrom3Hstandards.
20mg/kg of THC were observed in both vehicle and nandro-
lonepre-exposedmice.Two-wayANOVArevealedsignificant
2.4.2.NandroloneeffectsoncannabinoidCB1receptor
THC effect (F(3,87)¼23.60, P<0.001), but no nandrolone
activationofGTP-bindingprotein
TheprotocolusedwasbasicallythemethoddescribedbySimetal.(1995). effect (F(1,87)¼1.03, NS) and no THC/nandrolone interac-
Briefly, the brain structure were rinsed in assay buffer (50mM Tris, 3mM tion (F(3,87)¼1.44, NS). In the hot-plate test, significant
MgCl2,0.2mMEGTA,100mMNaCl,and0.5%bovineserumalbuminfatty antinociceptive effects of 10 and 20mg/kg were found inve-
acid-free,pH7.4)at25(cid:2)Cfor10min,thenpretreatedfor15minwithanex- hicle, but not in nandrolone pre-exposed mice. Two-way
cessconcentration(2mM)ofGDP(SigmaChemicalCo.,Madrid,Spain)inas-
ANOVA showed significant THC effect (F(3,87)¼8.22,
say buffer containing 0.04nM [35S]GTPgS (Amersham), 2mM GDP, and
5mM WIN 55 212-2 (Sigma Chemical Co., Madrid, Spain). Basal activity P<0.001), no nandrolone effect (F(1,87)¼0.07, NS) and
wasassessedintheabsenceofagonist,whereasnon-specificbindingwasmea- no THC/nandrolone interaction (F(3,87)¼1.18, NS).
suredinthepresenceof10mMunlabelledGTPgS.Sliceswererinsedtwicein Similarly, nandrolone co-administration did not modify
50mMTrisbuffer,pH7.4,at4(cid:2)Canddeionizedonceinwater,thendriedun- THC acute antinociceptive effects as evaluated in the tail-
derastreamofcooldryair.Autoradiogramsweregeneratedbyapposingthe
immersion and the hot-plate test (Table 1). In the tail-immer-
labelledtissuestofilm(KodakBiomaxMR)foraperiodof2daysanddevel-
oped for 4min at 20(cid:2)C. Developed films were analysed and quantified in sion test, significant antinociceptive effects of 5, 10 and
a computerized image analysis system (MCID, St. Catharines, Ontario, 20mg/kg of THC were observed in both vehicle and nandro-
Canada). lone co-treated mice. Two-way ANOVA revealed significant
THCeffect(F(3,105)¼26.11,P<0.001),butnonandrolone
2.5.Statistical analysis effect (F(1,105)¼0.03, NS) nor THC/nandrolone interaction
(F(3,105)¼0.40,NS).Inthehot-platetest,significantantino-
The data obtained for acute THC pharmacological responses, food and
ciceptiveeffectsof10and20mg/kgofTHCwereobservedin
THC effects in the place conditioning paradigm, rimonabant-precipitated
both vehicle and nandrolone co-treated mice. Two-way
THCwithdrawalandTHCeffectsonanxiety-likeresponsewereanalysedby
usingatwo-wayANOVAwithnandrolone/vehicleandTHC/vehicletreatment ANOVA revealed significant THC effect (F(3,104)¼11.25,
asfactorsofvariation.One-wayANOVAwasusedtorevealthemainTHCor P<0.001), but no nandrolone effect (F(1,104)¼3.81, NS)
nandroloneeffects,followedwhenappropriatebycorrespondingposthoccom- or THC/nandrolone interaction (F(3,104)¼0.22, NS).
parisons(Dunnett’stest)toindicategroupssignificantlydifferentfromcontrol.
Inaddition,individualcomparisonsoftimespentinthedrug-pairedcompart-
mentduringpreconditioningandtestphasesweremadeinplaceconditioning 3.1.2. THC hypothermic effects
experiments in each experimental group by using the paired two-tailed Chronic nandrolone pre-exposure during 14days and nan-
Student’st-test.
droloneco-administrationdidnotmodifyTHCacutehypother-
DatafromthestudyoftolerancetoTHCantinociception,hypothermiaand
miceffects(Table1).Similarsignificanthypothermiceffectsof
hypolocomotoreffectswereanalysedbyusingathree-wayANOVAwithtime
as the within subjects factor of variation and two between subjects factors THC were observed in vehicle and nandrolone exposed mice
792 E.Ce´le´rieretal./Neuropharmacology50(2006)788e806
Table1
EffectsofnandroloneontheacutepharmacologicaleffectsofD9-tetrahydrocannabinol(THC)
THCdoses Tail-withdrawal Hot-plate Rectal Totalhorizontal
latency(s) jumping(s) temperature locomotoractivity
((cid:2)C) (counts)
Pre-exposure
0mg/kg VEH 2.51(cid:1)0.12 47(cid:1)4 1.13(cid:1)0.59 265(cid:1)21
ND 2.50(cid:1)0.17 53(cid:1)12 1.81(cid:1)0.65 230(cid:1)23
5mg/kg VEH 2.70(cid:1)0.11 57(cid:1)5 (cid:4)1.46(cid:1)0.86 258(cid:1)25
ND 2.55(cid:1)0.16 66(cid:1)8 (cid:4)1.56(cid:1)0.74* 204(cid:1)32
10mg/kg VEH 4.84(cid:1)0.36** 108(cid:1)15** (cid:4)5.56(cid:1)1.08** 96(cid:1)12**
ND 3.73(cid:1)0.46* 92(cid:1)9 (cid:4)3.32(cid:1)1.04** 149(cid:1)23*
20mg/kg VEH 5.38(cid:1)0.37** 99(cid:1)14** (cid:4)7.52(cid:1)0.92** 104(cid:1)12**
ND 5.52(cid:1)0.36** 100(cid:1)16 (cid:4)7.87(cid:1)0.84** 94(cid:1)9**
Co-administration
0mg/kg VEH 2.22(cid:1)0.06 48(cid:1)3 1.24(cid:1)0.34 206(cid:1)13
ND 2.48(cid:1)0.13 58(cid:1)5 0.78(cid:1)0.26 213(cid:1)13
5mg/kg VEH 3.76(cid:1)0.50** 72(cid:1)11 (cid:4)3.14(cid:1)1.29** 135(cid:1)35*
ND 3.96(cid:1)0.39** 107(cid:1)17 (cid:4)4.83(cid:1)0.93** 106(cid:1)23**
10mg/kg VEH 4.89(cid:1)0.39** 107(cid:1)12** (cid:4)5.38(cid:1)0.86** 66(cid:1)11**
ND 4.39(cid:1)0.41** 124(cid:1)16** (cid:4)5.53(cid:1)0.90** 83(cid:1)17**
20mg/kg VEH 5.44(cid:1)0.44** 113(cid:1)19** (cid:4)6.71(cid:1)1.33** 97(cid:1)18**
ND 5.45(cid:1)0.60** 131(cid:1)20** (cid:4)7.87(cid:1)1.42** 86(cid:1)16**
VEH,vehicle;ND,nandrlone.*P<0.05,**P<0.05;Dunnet’stest,comparisonwithcontrolgroupreceivingvehicle.
underbothprotocols.Inthepre-exposureexperiment,two-way was administered using the two different protocols, i.e.,
ANOVA revealed significant THC effect (F(3,87)¼38.40, co-administration and pre-exposure.
P<0.001), but no nandrolone effect (F(1,87)¼0.92, NS)
or THC/nandrolone interaction (F(3,87)¼1.18, NS). In the 3.2.1. Tolerance to THC antinociceptive effects
co-administration experiment, two-wayANOVA revealedsig- Nandrolone pre-exposure and co-administration did not
nificantTHCeffect(F(3,105)¼32.39,P<0.001),butnonan- produceanychangeinthedevelopmentoftolerancetoantino-
drolone effect (F(1,105)¼1.15, NS) or THC/nandrolone ciception induced by chronic THC in the tail immersion
interaction(F(3,105)¼0.13,NS). (Fig. 1) and the hot-plate (data not shown) tests. Both proto-
cols of nandrolone treatment did not modify the nociceptive
3.1.3. THC hypolocomotor effects responses in these two tests in vehicle-treated mice. In THC
Chronic nandrolone pre-exposure during 14days and nan- chronically treated mice, tolerance to the antinociceptive
droloneco-administrationdidnotinfluenceTHCacuteeffects effectsofTHCinthetail-immersionandhot-platetestsdevel-
onhorizontallocomotoractivity(Table1).Significanthypolo- oped similarly in both vehicle and nandrolone pre-exposed
comotoreffectsofTHCwereobservedinvehicle-pre-exposed mice. Similar results were obtained in THC mice co-treated
mice and nandrolone pre-exposed mice under both protocols. with nandrolone. (see three-way ANOVA in Table 2).
In the pre-exposure experiment, two-way ANOVA revealed
significant THC effect (F(3,92)¼26.67, P<0.001), but no 3.2.2. Tolerance to THC hypothermic effects
significant effect of nandrolone (F(1,92)¼0.66, NS) or Nandrolone pre-exposure and co-administration did not
THC/nandrolone interaction (F(3,92)¼0.07, NS). In the co- produce any change in the development of tolerance to the
administration experiment, two-way ANOVA also revealed hypothermic effects of THC (Fig. 1). Both protocols of nan-
significant THC effect (F(3,78)¼26.06, P<0.001), but no drolone treatment did not modify body temperature in vehi-
significant effect of nandrolone (F(1,78)¼0.09, NS) or cle-treated mice. In THC chronically treated mice, tolerance
THC/nandrolone interaction (F(3,78)¼0.52, NS). to the hypothermic effects of THC developed similarly in
bothvehicleandnandrolonepre-exposedmice.Similarresults
3.2.Effectsofnandrolonetreatmentsonthedevelopment were obtained in THC mice co-treated with nandrolone (see
of tolerance to THC effects three-way ANOVA in Table 2).
Changes in nociceptive thresholds, rectal temperature and 3.2.3. Tolerance to THC hypolocomotor effects
locomotor activity were evaluated every day during chronic Nandrolone pre-exposure and co-administration did not
administration of THC (20mg/kg, i.p., twice a day for produceanychangeinthedevelopmentoftolerancetothehy-
5days) or vehicle. Nandrolone (15mg/kg, i.m.) or vehicle plocomotor effects of THC (Fig. 1). Both protocols of
E.Ce´le´rieretal./Neuropharmacology50(2006)788e806 793
A: Nandrolone pre-exposure B: Nandrolone co-administration
VEH-VEH
7 7 ND-VEH
** ** VEH-THC
awal (s) 456 ** **** ** ** * 456 ** **** ** ** *N*D-THC
hdr 3 ** ** 3 ** **
wit 2 2
ail 1 1
T
0 0
0 1 2 3 4 5 0 1 2 3 4 5
Days Days
4 4
mperaturesal value) --4202 ** *** --4202 **** **** *
ea
ectal t% of b --86 ** ** --86 **
R( -10 -10 **
**
-12 -12
1 2 3 4 5 1 2 3 4 5
Days Days
300 ** 300
vity 250 250 **
actis) 200 ** 200 **
motor (count 110500 *** ** ** ** 110500 * **
o *
c **
o 50 50 **
L
0 0
0 1 2 3 4 5 0 1 2 3 4 5
Days Days
Fig.1.EffectsofnandrolonetreatmentsonthedevelopmentoftolerancetoTHC-inducedantinociceptive,hypothermicandhypolocomotoreffects.THC(20mg/
kg,i.p.)waschronicallyadministeredtwiceadayduring5days.Tolerancedevelopmentwasevaluatedinmicepre-exposed(A)toorco-treated(B)withnan-
drolone(ND)orvehicle(VEH).Locomotoractivitywasrecorded20minaftereachmorningTHCinjectionduring10min.Antinociceptiveeffectswereevaluated
30minaftereachmorningTHCinjectionbyusingthetail-immersiontest.Therectaltemperaturewasmeasuredjustbeforeand45minaftereachmorningTHC
injection.Thenumberofmicepergroupinthenandrolonepre-exposureexperimentswas10e13.Thenumberofmicepergroupinthenandroloneco-treatment
experimentswas11e15.Dataareexpressedasmean(cid:1)SEM.*P<0.05,**P<0.01(one-wayANOVA,comparisonwiththerespectivebasalvalue).
nandrolonetreatmentdidnotmodifylocomotoractivityinve- injection of rimonabant in vehicle control mice co-treated or
hicle-treatedmice.InTHCchronicallytreatedmice,tolerance pre-exposed to nandrolone. Rimonabant injection in chronic
to the hypolocomotor effects of THC developed similarly in THC treated mice with vehicle pre-exposure precipitated
bothvehicleandnandrolonepre-exposedmice.Similarresults a withdrawal syndrome manifested by the presence of wet
were obtained in THC mice co-treated with nandrolone (see dog shake (one-way ANOVA, F(1,24)¼15.07, P<0.01),
three-way ANOVA in Table 2). front paw tremor (F(1,24)¼33.09, P<0.001), ptosis
(F(1,24)¼29.25, P<0.001) and a significant global with-
3.3. Effects of nandrolone treatments on drawal score (F(1,24)¼62.53, P<0.01), as revealed by
rimonabant-precipitated THC withdrawal syndrome one-way ANOVA (Fig. 2). The intensity of THC withdrawal
was increased in nandrolone pre-exposed mice (Fig. 2) (see
THC withdrawal was precipitated by the administration of two-way ANOVA in Table 3). One-way ANOVA revealed
the selective CB1 receptor antagonist, rimonabant (10mg/kg a significant increase of sniffing (F(1,21)¼4,61, P<0.05)
i.p.), in mice receiving chronic administration of THC and global withdrawal score (F(1,21)¼5.12, P<0.05) in
(20mg/kg, i.p., twice daily during 6days) or vehicle. No sig- micepre-exposedtonandroloneincomparisonwiththegroup
nificant signs of withdrawal were observed in any group of pre-exposed to vehicle.
mice during the 15min behavioural observation performed Incontrast,theintensityofTHCwithdrawalwasnotmodified
beforetheadministrationofrimonabant.Moreover,nobehav- by the co-administration of nandrolone (Fig. 2). Rimonabant-
ioural manifestations of withdrawal were observed after the precipitated THC withdrawal was manifested in vehicle
794 E.Ce´le´rieretal./Neuropharmacology50(2006)788e806
Table2 3.4.1. THC rewarding properties
NandroloneeffectsontolerancetoD9-tetrahydrocannabinol(THC)pharmaco- In the nandrolone pre-exposure experiment, two-way AN-
logicaleffects
OVA revealed no significant THC (F(1,56)¼1.48, NS) and
Nandrolonepre-exposure Nandroloneco-administration nandrolone (F(1,56)¼0.80, NS) effects, but significant
F P F P THC/nandrolone interaction (F(1,56)¼4.64, P<0.05). Re-
THCantinociceptiveeffects(tail-flicktest) wardingeffectsofTHC(1mg/kg)wereobservedinmicepre-
THC (1,43)¼63.36 <0.001 (1,48)¼101.25 <0.001 viously receiving intramuscular vehicle for 14days (Fig. 3A)
Time (4,172)¼15.11 <0.001 (4,192)¼15.39 <0.001 as revealed by the score values (one-way ANOVA;
ND (1,43)¼0.03 NS (1,48)¼0.36 NS
F(1,29)¼5.37, P<0.05) and the increase in the time spent
THC(cid:3)time (4,172)¼14.41 <0.001 (4,192)¼16.29 <0.001
inthedrug-pairedcompartmentduringthetestvs.theprecon-
THC(cid:3)ND (1,43)¼0.14 NS (1,48)¼0.22 NS
ND(cid:3)time (4,172)¼0.08 NS (4,192)¼0.09 NS ditioning phase (Student’s t-test, t(1,14)¼(cid:4)3.27, P<0.01).
THC(cid:3)ND(cid:3) (4,172)¼1.02 NS (4,192)¼0.13 NS On the contrary, THC did not induce rewarding effects in
time mice pre-exposed to nandrolone, as revealed by the similar
timespentinthedrug-pairedcompartment duringtheprecon-
THCantinociceptiveeffects(hot-platetest)
ditioningandtestphase(Student’st-test,t(1,14)¼0.193,NS)
THC (1.43)¼7.10 <0.05 (1,47)¼13.48 <0.01
Time (4,172)¼42.53 <0.001 (4,188)¼163.17 <0.001 and the scorevalues (one-way ANOVA, F(1,29)¼0.47, NS).
ND (1,43)¼0.36 NS (1,47)¼2.48 NS Significant differences were also observed in THC-treated
THC(cid:3)time (4,172)¼7.58 <0.01 (4,188)¼3.17 NS mice when compared with vehicle and nandrolone pre-
THC(cid:3)ND (1,43)¼0.19 NS (1,47)¼0.35 NS
exposed mice (one-way ANOVA, F(1,29)¼4.87, P<0.05).
ND(cid:3)time (4,172)¼0.73 NS (4,188)¼0.01 NS
The influence of chronic nandrolone co-administration on
THC(cid:3)ND(cid:3) (4,172)¼1.04 NS (4,188)¼0.02 NS
time THCrewardingeffects could notbeassessed dueto the inter-
ference of the previous intramuscular injection. Two-way
THChypothermiceffects ANOVA revealed no significant THC (F(1,53)¼1.81, NS)
THC (1.43)¼74.85 <0.001 (1,47)¼87.73 <0.001
and nandrolone (F(1,53)¼1.20, NS) effects, and no signifi-
Time (4,172)¼25.10 <0.001 (4,188)¼21.74 <0.001
cant THC/nandrolone interaction (F(1,53)¼0.01, NS). THC
ND (1,43)¼0.04 NS (1,47)¼6.31 <0.05
THC(cid:3)time (4,172)¼36.79 <0.01 (4,188)¼21.48 <0.001 (1mg/kg, s.c.) did not induce conditioned place preference
THC(cid:3)ND (1,43)¼2.61 NS (1,47)¼1.07 NS when mice received an intramuscular injection of vehicle or
ND(cid:3)time (4,172)¼0.76 NS (4,188)¼0.44 NS nandrolone 1h before the conditioning session (Fig. 3B), as
THC(cid:3)ND(cid:3) (4,172)¼1.55 NS (4,188)¼1.06 NS
revealedbythesimilartimespentinthedrug-pairedcompart-
time
mentonthepreconditioningandtestphaseintheTHC-treated
THChypothermiceffects mice(Student’st-test,t(1,11)¼0.42,NS,inthevehiclegroup
THC (1.43)¼46.84 <0.001 (1,47)¼28.36 <0.001 andt(1,11)¼0.68,NSinthenandrolonegroup)andscoreval-
Time (4,172)¼41.88 <0.001 (4,188)¼36.01 <0.001 ues (one-way ANOVA, F(1,25)¼0.37, NS, in the vehicle
ND (1,43)¼1.13 NS (1,47)¼0.56 NS
group and one-way ANOVA, F(1,25)¼0.19, NS, in the nan-
THC(cid:3)time (4,172)¼7.8 <0.001 (4,188)¼5.60 <0.001
drolone group). On the contrary, a significant conditioned
THC(cid:3)ND (1,43)¼3.62 NS (1,47)¼0.21 NS
ND(cid:3)time (4,172)¼0.51 NS (4,188)¼1.75 NS place preference was observed in a positive control group of
THC(cid:3)ND(cid:3) (4,172)¼1.63 NS (4,188)¼0.31 NS mice receiving THC alone under the same experimental con-
time ditions without any intramuscular administration, as revealed
Three-wayANOVAwithTHCandnandrolone(ND)(betweensubjects)and by the increase in the time spent in the drug-paired compart-
time(withinsubjects)asfactorsofvariations.NS,notsignificant. ment during the test phase (Student’s t-test, t(1,9)¼(cid:4)2.39,
P<0.05), and the score values (one-way ANOVA,
co-administrated mice by the presence of wet dog shake (one- F(1,25)¼5.06, P<0.05).
way ANOVA, F(1,25)¼30.09, P<0.001), front paw tremor
(F(1,25)¼25.76, P<0.001), sniffing (F(1,25)¼4.72, P< 3.4.2. THC aversive properties
0.05), ptosis (F(1,25)¼40.76, P<0.001) and a significantly In the nandrolone pre-exposure experiment, two-way
globalwithdrawalscore(F(1,25)¼40.94,P<0.01),asrevealed ANOVA revealed significant THC effect (F(1,51)¼13.68,
by one-way ANOVA. Two-way ANOVA (Table 3) revealed P<0.01), but no effect of nandrolone (F(1,51)¼0.76, NS)
similar manifestations of all the withdrawal signs in chronic or THC/nandrolone interaction (F(1,51)¼0.05, NS). Signifi-
THCtreatedmiceco-administeredwithvehicleornandrolone. cant aversive effects of THC (5mg/kg) were observed in
mice previously receiving intramuscular vehicle for 14days
(Fig. 3A), as revealed by the score values (one-way ANOVA,
3.4.EffectsofnandrolonetreatmentsonTHCrewarding F(1,27)¼8.87, P<0.01) and the decrease in the time spent
and aversive properties inthedrug-pairedcompartmentduringthetestvs.theprecon-
ditioning phase (Student’s t-test, t(1,12)¼(cid:4)3.25, P<0.01).
The influence of nandrolone exposure on THC rewarding Nandrolone pre-exposure did not modify THC aversive ef-
andaversiveeffectswasinvestigatedbyusingtheplacecondi- fects, as revealed by the score values (one-way ANOVA,
tioned paradigm (Fig. 3). F(1,27)¼0.41, NS) and the decrease in the time spent in
E.Ce´le´rieretal./Neuropharmacology50(2006)788e806 795
A: Nandrolone pre-exposure
kes 60 mor **
Wet dog sha 24000 VEH *T*H*C* Front paw tre 1200000 VEH *T*HC al score 111024000 VNEDH *#*
w
g 12 *#* 6 **** withdra 6800 **
Sniffin 48 Ptosis 24 Global 2400
0 0 0
VEH THC VEH THC VEH THC
B: Nandrolone co-administration
kes 60 **** mor ****
Wet dog sha 24000 VEH THC Front paw tre 1200000 VEH THC al score 111024000 VNEDH ** **
w
a 80
12 6 **** hdr 60
Sniffing 48 *** Ptosis 24 obal wit 2400
Gl
0 0 0
VEH THC VEH THC VEH THC
Fig.2.Effectsofnandrolonetreatmentsontherimonabant-precipitatedTHCwithdrawalsyndrome.AbstinenceisprecipitatedbytheadministrationoftheCB1
receptorantagonistrimonabant(10mg/kg,i.p.)inmicereceivingachronictreatmentofTHC(20mg/kg,i.p.),twiceadayduring6days.Micewerepre-exposed
(A)toorco-treated(B)withnandrolone(ND)orvehicle(VEH).Somaticsignsofwithdrawalwereobservedfor45minimmediatelyaftertherimonabantad-
ministration.Theglobalwithdrawalscorewascalculatedforeachanimalbygivingeachindividualsigna relativeweight.Thenumberofmicepergroupin
thenandrolonepre-exposureexperimentswas10e13.Thenumberofmicepergroupinthenandroloneco-treatmentexperimentswas10e15.Dataareexpressed
asmean(cid:1)SEM.*P<0.05,**P<0.01(one-wayANOVA,comparisonwiththerespectivebasalvalue)and#P<0.05(one-wayANOVA,VEH-vsND-treated
mice).
the drug-paired compartment during the test vs. precondition- couldnotbeassessedduetotheinterferenceofthepreviousin-
ing phase (Student’s t-test, t(1,12)¼4.55, P<0.01). No dif- tramuscular injection. Two-way ANOVA revealed no signifi-
ferences were observed in the aversive effects of THC when cant THC (F(1,65)¼2.19, NS) and nandrolone (F(1,65)¼
compared vehicle and nandrolone pre-exposed mice (one- 0.27, NS) effects, and no significant THC/nandrolone interac-
way ANOVA, F(1,25)¼0.31, NS). tion(F(1,65)¼1.17,NS).THC(5mg/kg,s.c.)didnotinduce
Similartothepreviousexperiment,theinfluenceofchronic conditionedplaceaversionwhenmicereceivedanintramuscu-
nandrolone co-administration on THC aversive properties lar injection of vehicle or nandrolone 1h before the
Table3
Nandroloneeffectsonrimonabant-precipitatedD9-tetrahydrocannabinol(THC)withdrawal
THC P ND P Interaction P
Nandrolonepre-exposure
Wetdogshakes (1,43)¼43.24 <0.001 (1,43)¼1.55 NS (1,43)¼2.99 NS
Frontpawtremor (1,43)¼68.20 <0.001 (1,43)¼4.91 <0.05 (1,43)¼4.58 <0.05
Sniffing (1,43)¼12.31 <0.01 (1,43)¼5.28 <0.05 (1,43)¼5.28 <0.05
Ptosis (1,43)¼66.32 <0.001 (1,43)¼3.94 NS (1,43)¼0.01 NS
Globalwithdrawalscore (1,43)¼109.13 <0.001 (1,43)¼6.31 <0.05 (1,43)¼5.49 <0.05
Nandroloneco-administration
Wetdogshakes (1,46)¼47.82 <0.001 (1,46)¼0.04 NS (1,46)¼0.07 NS
Frontpawtremor (1,46)¼41.15 <0.001 (1,46)¼0.07 NS (1,46)¼0.10 NS
Sniffing (1,46)¼5.81 <0.05 (1,46)¼3.01 NS (1,46)¼3.01 NS
Ptosis (1,46)¼46.17 <0.001 (1,46)¼0.16 NS (1,46)¼1.08 NS
Globalwithdrawalscore (1,46)¼60.45 <0.001 (1,46)¼0.09 NS (1,46)¼0.12 NS
Two-wayANOVAwithTHCandnandrolone(ND)(betweensubjects)asfactorsofvariations.NS,notsignificant.
796 E.Ce´le´rieretal./Neuropharmacology50(2006)788e806
A: Nandrolone pre-exposure B: Nandrolone co-administration
*
THC (1 mg/kg)
#
120 * 120
80 80
40 40
e
or 0 0
c
S
-40 -40
-80 -80
VEH ND VEH ND VEH ND VEH ND _
-120 VEH VEH THC THC -120 VEH VEH THC THC THC
THC (5mg/kg)
120 120
80 80
40 40
e
cor 0 0
S
-40 -40
-80 -80
-120 * -120
**
VEH ND VEH ND VEH ND VEH ND *_
VEH VEH THC THC VEH VEH THC THC THC
Food #
*
160 160
*
120 120
80 80
e
or 40 40
c
S
0 0
_
VEH ND VEH ND VEH ND VEH ND
-40 _ _ FD FD -40 _ _ FD FD FD
-80 -80
Fig.3.EffectsofnandrolonetreatmentsonrewardingandaversivepropertiesofTHCandfood.Thedosesof1and5mg/kg(i.p.)ofTHCwereusedtoinduce
conditionedplacepreferenceandaversion,respectively,inmicepre-exposedto(A)orco-treated(B)withnandrolone(ND)orvehicle(VEH).Food(FD)-induced
conditionalplacepreferencewasassessedonlightlyfooddeprivedmice.Ascorewascalculatedforeachmouseasthedifferencebetweenthepost-conditioning
and the pre-conditioning time spent in the drug- or food-paired compartment. The number of mice per group was 10e15. *P<0.05, **P<0.01 (one-way
ANOVA,comparisonwiththerespectivebasalvalue)and#P<0.05(one-wayANOVA,VEH-vsND-treatedmice).
conditioningsession(Fig.3B),asrevealedbythesimilartime aversion was observed in a positive control group receiving
spentinthedrug-pairedcompartmentduringtheprecondition- THC alone under the same experimental conditions without
ingandtestphase(Student’st-test,t(1,14)¼(cid:4)0.60,NS,inthe any intramuscular administration, as revealed by the increase
vehiclegroupandt(1,14)¼0.88,NSinthenandrolonegroup) in the time spent in the drug-paired compartment during
andscorevalues(one-wayANOVA,F(1,29)¼0.15,NS,inthe the test phase (Student’s t-test, t(1,9)¼(cid:4)2.81, P<0.05)
vehicle group and F(1,29)¼0.21, NS, in the nandrolone and the score values (one-way ANOVA, F(1,29)¼4.85,
group). On the contrary, a significant conditioned place P<0.05).
E.Ce´le´rieretal./Neuropharmacology50(2006)788e806 797
3.5. Effects of nandrolone treatments on the rewarding ANOVA. Anxiolytic-like effects of THC (0.2mg/kg) were
properties of food also observed in the lit/dark box (Fig. 4A and see Table 6
for two-way ANOVAs). THC increased the percentage of en-
In mice previously receiving intramuscular vehicle for triesinthelitcompartment(F(1,35)¼5.10,P<0.05)andthe
14days (Fig. 3A), two-way ANOVA revealed no significant entriesinthedistalareaofthiscompartment(F(1,35)¼21.63,
food (F(1,40)¼2.87, NS) and nandrolone (F(1,40)¼2.90, P<0.001), and decreased the entries in its proximal area
NS) effects, but a significant food/nandrolone interaction (F(1,35)¼21.03, P<0.001), as revealed by one-way
(F(1,40)¼4.35, P<0.05). Significant conditioned place ANOVA. THC also increased the percentage of squares
preference to food was observed in these mice, as revealed crossed in the lit compartment (F(1,35)¼42.22, P<0.001).
by the score values (one-way ANOVA, F(1,21)¼0.29, NS) The anxiolytic-like effects of THC (0.2mg/kg) were not re-
andtheincreaseinthe timespent inthe food-pairedcompart- vealed in the elevated plus-maze in our experimental condi-
ment during the test vs. preconditioning phase (Student’s tions (Fig. 6A and see Table 7 for two-way ANOVA). In
t-test, t(1,9)¼3.18, P<0.05). On the contrary, rewarding mice pre-treated with nandrolone, THC (0.2mg/kg) did not
effectsoffoodwereabolishedinmicepre-exposedtonandro- produce any anxiolytic-like effect in the open-field
lone, as revealed by the similar time spent in the food-paired (Fig. 5A), lit/dark box (Fig. 4A) and elevated plus-maze
compartment during the test vs. preconditioning phase (Stu- (Fig. 6A). One-way ANOVA revealed significant differences
dent’s t-test, t(1,10)¼0.26, NS) and the score values (one- in THC effects between vehicle and nandrolone pre-treated
way ANOVA, F(1,22)¼0.09, NS). Significant differences mice in the open-field (number of rearing: F(1,24)¼44.61,
were also observed between vehicle and nandrolone pre- P<0.001; percentage of squares crossed in the central area:
exposedmiceconditionedwithfood(F(1,20)¼7.31,P<0.05). F(1,24)¼25.94, P<0.001) and the lit/dark box (entries
Similar to the previous experiment, food did not induce into the proximal area of the lit compartment:
conditionedplacepreferenceinmicereceivinganintramuscu- F(1,38)¼40.36, P<0.001; entries into the distal area of
larinjectionofvehicleornandrolone1hbeforethecondition- the lit compartment: F(1,38)¼32.71, P<0.001, and squares
ing session (Fig. 3B), as revealed by the similar time spent in crossedintothelitcompartment:F(1,38)¼30.41,P<0.001).
the drug-paired compartment on the preconditioning and test The influence of chronic nandrolone co-administration on
phase (Student’s t-test, t(1,11)¼(cid:4)0.51, NS, in the vehicle THC anxiolytic-like effects could not be assessed due to the
group and t(1,12)¼(cid:4)0.17, NS in the nandrolone group), interference of the previous intramuscular injection (Figs.
and the score values (one-way ANOVA, F(1,25)¼0.12, 4Be6B and Tables 5e7 for two-way ANOVA). Indeed,
NS). Two-way ANOVA revealed no significant food THC (0.2mg/kg) did not produce any anxiolytic-like effects
(F(2,54)¼1.30,NS)andnandrolone(F(1,54)¼0.11,NS)ef- inanyofthe behaviouraltests whenmice receiveda previous
fects, and no food/nandrolone interaction (F(1,54)¼0.03, intramuscular injection of vehicle.
NS). On the contrary, a significant place preference was ob-
servedinapositivecontrolgroupofmicereceivingfoodunder 3.6.2.Influenceofnandroloneontheanxiogenic-likeeffects
the same experimental conditions without any intramuscular of THC (7.5mg/kg)
administration, as revealed by the significant increase in the In mice pre-treated with vehicle, the dose of 7.5mg/kg of
time spent in the drug-paired compartment during the test THC produced anxiogenic-like effects in the open-field, lit/
phase (Student’s t-test, t(1,10)¼(cid:4)2.88, P<0.05) and the darkboxandelevatedplus-maze(Figs.4Ae6AandseeTables
score values (one-way ANOVA, F(1,25)¼4.92, P<0.05). 5e7for two-way ANOVA). Inthe open-field, THC decreased
the number of rearing (F(1,24)¼19.00, P<0.001) and the
3.6. Effects of nandrolone treatments on the THC percentage of squares crossed into the central area
anxiolytic- and anxiogenic-like responses (F(1,24)¼22.64, P<0.001), as revealed by one-way
ANOVA. In the elevated plus-maze, THC decreased the per-
The influence of nandrolone on THC-induced changes in centage of time spent in the open arms (one-way ANOVA,
anxiety-like responses was evaluated in the lit/dark box, F(1,29)¼4.95, P<0.05). In the lit/dark box test, THC in-
open-field and elevated plus-maze (Figs. 4e6) and the global creased the percentage of entries in the proximal area of the
results obtainedare summarized inTable 4.Both protocols of lit compartment (F(1,36)¼9.80, P<0.01), and decreased
nandrolonetreatmentshadnointrinsiceffectwheninjectedin the entries in its distal area (F(1,36)¼9.57, P<0.01) and
combination with vehicle in any of the parameters studied. the squares crossed in the lit compartment (F(1,36)¼8.49,
P<0.01), as revealed by one-way ANOVA. In mice pre-
3.6.1. Influence of nandrolone on the anxiolytic-like effects treated with nandrolone, THC (7.5mg/kg) produced similar
of THC (0.2mg/kg) anxiogenic-like effects in the open-field, the lit/dark box and
In mice pre-treated with vehicle, the dose of 0.2mg/kg of theelevatedplus-maze.Intheopen-field,one-wayANOVAre-
THC produced anxiolytic-like effects in the open-field vealed a decrease in the number of rearing (F(1,22)¼17.17,
(Fig. 5A and see Table 5 for two-way ANOVA). THC in- P<0.001) and the squares crossed into the central area
creased the number of rearing (F(1,24)¼44.10, P<0.001) (F(1,22)¼29.94, P<0.001). In the elevated plus-maze,
and the percentage of squares crossed in the central area one-way ANOVA showed a decrease in the time spent in
(F(1,24)¼20.07, P<0.001), as revealed by one-way the open arms (F(1,29)¼14.10, P<0.01) and the total