Chronic Exposure to Anabolic Androgenic Steroids Alters Neuronal Function in the Mammalian ...

13 Pages·2009·0.85 MB·English

12484•TheJournalofNeuroscience,October7,2009•29(40):12484–12496
Behavioral/Systems/Cognitive
Chronic Exposure to Anabolic Androgenic Steroids Alters
Neuronal Function in the Mammalian Forebrain via
Androgen Receptor- and Estrogen Receptor-Mediated
Mechanisms
CarlosA.A.Penatti,DonnaM.Porter,andLeslieP.Henderson
DepartmentofPhysiology,DartmouthMedicalSchool,Hanover,NewHampshire03755
Anabolicandrogenicsteroids(AAS)canpromotedetrimentaleffectsonsocialbehaviorsforwhichGABAtypeA(GABA )receptor-
A
mediatedcircuitsintheforebrainplayacriticalrole.WhileallAASbindtoandrogenreceptors(AR),theymayalsobearomatizedto
estrogensandthuspotentiallyimparteffectsviaestrogenreceptors(ER).Chronicexposureofwild-typemalemicetoacombinationof
chemicallydistinctAASincreasedactionpotential(AP)frequency,selectiveGABA receptorsubunitmRNAs,andGABAergicsynaptic
A
currentdecayinthemedialpreopticarea(mPOA).ExperimentsperformedwithpharmacologicalagentsandinAR-deficientTfmmutant
micesuggestthattheAAS-dependentenhancementofGABAergictransmissioninwild-typemiceisAR-mediated.InAR-deficientmice,
theAASeliciteddramaticallydifferenteffects,decreasingAPfrequency,spontaneousIPSCamplitudeandfrequencyandtheexpression
ofselectiveGABA receptorsubunitmRNAs.Surprisingly,intheabsenceofARsignaling,thedataindicatethattheAASdonotactasER
A
agonists,butrathersuggestanovelinvivoactioninwhichtheAASinhibitaromataseandimpairendogenousERsignaling.Theseresults
showthattheAAShavethecapacitytoalterneuronalfunctionintheforebrainviamultiplesteroidsignalingmechanismsandsuggestthat
effectsofthesesteroidsinthebrainwilldependnotonlyonthebalanceofAR-versusER-mediatedregulationfordifferenttargetgenes,
butalsoontheabilityofthesedrugstoaltersteroidmetabolismandthustheendogenoussteroidmilieu.
Introduction shown to both advance and retard pubertal onset in females,
Anabolicandrogenicsteroids(AAS)aresyntheticderivativesof enhanceanddiminishlibidoinbothsexes,andsuppressrepro-
testosterone designed for therapeutic uses, but now predomi- ductivecompetenceinbothsexes(FrankeandBerendonk,1997;
nantlyself-administeredtoenhanceperformanceorbodyimage ClarkandHenderson,2003;Clarketal.,2006).AASuseisalso
(KochakianandYesalis,2000;Llewellyn,2007).Humansadmin- associated with both enhanced and diminished aggression and
isterAASincomplexregimescharacterizedbyconcurrentand anxiety (Clark and Henderson, 2003; Trenton and Currier,
prolongeduseofmultipleAASatdosesthatresultinsupraphysi- 2005).BothAR-andER-mediatedsignalingarecriticallyimpor-
ologicallevelsofthesesteroids,and(cid:1)100AASareavailablethat tantwithrespecttotheactionsofgonadalsteroidsonthepro-
varywidelyintheirchemicalpropertiesandtheirmetabolicfates ductionandcomplexityofthesebehaviors.Forexample,inmale
(Llewellyn,2007).Importantly,althoughallAASandAASme- rodents,thefullrepertoireofsexualbehaviorsreflectstheactions
tabolites bind to the classical androgen receptor (AR), many, ofbothandrogensandestrogens(Hulletal.,2002).Similarly
uponaromatization,mayalsoexertphysiologicaleffectsviaclas- both androgens and estrogens can activate offensive aggres-
sical estrogen receptor (ER(cid:1)and (cid:2)) pathways (Basaria et al., sioninadultmalemice(Simon,2002;Matsumotoetal.,2003;
2001;Shahidi,2001;Clarketal.,2006). ScordalakesandRissman,2003;Satoetal.,2004).
While administered for their anabolic actions, AAS use has Expressionofthesesteroid-sensitivebehaviorsisalsodepen-
also been strongly correlated with untoward actions on social dentonGABAAreceptor-mediatedtransmissioninregionsofthe
behaviors.SpecificeffectsvarywiththeregimeofAASadminis- forebrain including the mPOA (Blaustein and Erskine, 2002;
tration(duration,doseanddrugcombinations)andwithsexand Hulletal.,2002;Miczeketal.,2002;SewardsandSewards,2002;
ageofthesubject.Dependingonthesevariables,AAShavebeen Goodson,2005;Veeningetal.,2005).Thecentralportionofthe
mPOA is characterized by a dense population of neurons that
are nearly all GABAergic (Gao and Moore, 1996; Sagrillo and
ReceivedJune30,2009;revisedAug.25,2009;acceptedAug.26,2009.
ThisworkwassupportedbytheNationalInstitutesofHealth(GrantsDA18255andDA14137).WethankDrs.Ann Selmanoff,1997)andGABA-responsive(Penattietal.,2005;this
ClarkandJosephOberlanderandMr.BenjaminSolomonforcriticalreviewofthismanuscriptandDr.BethCostinefor study).ThesecellsalsoexpresshighlevelsofAR(Luetal.,1998;
statisticalassistance. Shahetal.,2004)andER(cid:1),lowerbutappreciablelevelsofER(cid:2)
Correspondence should be addressed to Leslie P. Henderson at the above address. E-mail: leslie.
(Mitraetal.,2003;Nomuraetal.,2003;Kudwaetal.,2004),and
[email protected].
highlevelsofaromatase(Foidartetal.,1995).GABAergictrans-
DOI:10.1523/JNEUROSCI.3108-09.2009
Copyright©2009SocietyforNeuroscience 0270-6474/09/2912484-13$15.00/0 missioninthemPOAisregulatedbygonadalsteroids(Herbison,
Penattietal.•AASEffectsintheForebrain J.Neurosci.,October7,2009•29(40):12484–12496•12485
1997)andtheAAS(Hendersonetal.,2006).ThediversityofAAS kg/d)(Yueetal.,1995),or17(cid:2)-estradiol(5(cid:3)gpermouse)(Bakkeretal.,
effectsonsocialbehaviorsislikelytoreflectacomplexbalance 2004)(forreview,seeCorniletal.,2006)wasinjectedintomicefor6d
andinterplaybetweenAR-andER-mediatedactionsonGABAergic perweekfor6weekseitheraloneorinconjunctionwiththeAASmixture
andotherneuralsignalingpathwaysinmPOAandconnected at concentrations indicated above. For physiological experiments in
which multiple cohorts were examined, if there was not a significant
regions. Here, we have taken advantage of mutant mice and
differencewithinatreatmentgroup,thedatafrommultiplecohortswere
pharmacologicalmanipulationstoassesstherolesofARver-
combined.Differentcohortsofanimalswereusedforeachassay(i.e.,
sus ER in mediating the effects of the AAS within this key
electrophysiology,immunocytochemistry,realtimePCR,ELISAs,and
forebrainregion. Westernblots).ForER(cid:1)Westerns,mPOAsamplesfromoneTfmand
onewild-typemousewerelostatthetimeofassay(thus,n(cid:5)7subjects).
MaterialsandMethods ForELISAs,mPOAtissuefromindividualanimalswaspooledinsome
Animals.AsinglepointmutationintheNterminusoftheARgeneinthe cases,andnvaluesreflectthenumberofdeterminations,ratherthan
testicularfeminization(Tfm)mouseresultsintheproductionofatrun- individualanimals(seebelow).
cated and nonfunctional receptor devoid of both DNA- and steroid- Tissue analyses. All electrophysiological analyses were made from
bindingdomains(Charestetal.,1991;Gasparetal.,1991;Olsen,1992; thecentralregionofthemPOAcorrespondingtothedorsalaspect
CouseandKorach,1998).WhilerecognizingthatTfmmicemakeatrun- ofthemedialpreopticnucleus(MPN),MPN-medialandencompass-
cated AR, we have called these animals functionally “AR-deficient” ingtheMPN-central,asdefinedbyFranklinandPaxinos(1997)(Penatti
ratherthan“androgen-insensitive,”sinceitisnotknownhowandrogen etal.,2005).FormRNA,steroidandproteinassessments,dissections
signalingmechanismsatnonclassical(e.g.,membrane)ARareaffectedin weremadefromaregionapproximatingtheMPN,butlikelytocontain
Tfmmiceandallostericmodulationofionchannelsbyandrogensoccurs surroundingcellsinthemPOAregion.Throughoutthetext,wereferto
intheseanimals(e.g.,seeJorgeetal.,2002).Becausethemutationis electrophysiologicalrecordingsasmadefromtheMPNandcellbiologi-
X-linked,allTfmmicegeneratedbyconventionalbreedingmethodsare calanalysesasmadefromthemPOAtoreflectthefactthattissuesamples
infertileandgeneticallymale(CouseandKorach,1998).Matingsoforig- werelikelytoincludethismoreextensiveregion.
inalbreederpairsofheterozygousAw(cid:2)J/Aw(cid:2)JEdaTa(cid:2)6J(cid:3)/(cid:3)ArTfmfe- RNAextractionandreversetranscriptioncoupledwithquantitativereal
males and wild-type Tabby Aw(cid:2)J/Aw(cid:2)J EdaTa(cid:2)6J (cid:3)/Y males obtained timePCR.Tissuedissectedfromcoronalslicesasdescribedabovewas
from Jackson Laboratories (stock # 001809) have generated a long- storedintheRNAstabilizationsolution,RNAlater(Ambion)at(cid:2)20°C.
standingbreedingcolonyatourfacilitythathasprovidedboththeAR- Total RNA was extracted according to manufacturer’s protocol for
deficientTfm(Aw(cid:2)J/Aw(cid:2)J(cid:3)ArTfm/Y)malesandwild-typeTabbymale RNAqueous-4PCR kit (Ambion). Briefly, tissue was added to 200 (cid:3)l
littermatesusedinthisstudy.OffspringweregenotypedbyPCRaccord- lysis/bindingbufferandhomogenized.Anequalvolumeof64%EtOH
ingtoScordalakesetal.(2002).Allmicewerehousedinatemperature- wasaddedandthesamplevortexed.Thelysate/ethanolmixwasapplied
controlledand12/12hon/offlightcyclefacilitywithlightsonstartingat toanRNAqueousfilter(suppliedinthekit)andcentrifugedfor1minat
7:00 A.M. All animal care procedures and treatment paradigms were 12,000rpm.Theflowthroughwasdiscarded,thecartridgewashedsev-
approvedbytheInstitutionalAnimalCareandUseCommitteeatDart- eraltimesandRNAelutedwith50(cid:3)lofhotelutionbuffer.TheRNAwas
mouthandareinagreementwiththeguidelinesandrecommendations treatedwith2unitsDNase1(suppliedinthekit)for30minat37°Cto
oftheNationalInstitutesofHealthandAmericanVeterinaryMedical removeanycontaminatinggenomicDNA.DNasewasinactivatedbythe
Association. additionof0.1volumeDNaseInactivationReagent,incubatedatroom
Drugtreatmentparadigms.Adult((cid:4)56dold)malemicewereinjected temperature(20(cid:2)22°C)for2min,centrifuged,andtheDNase-freeRNA
intraperitoneally with a combination of three AAS that represent the supernatantcollected.TheconcentrationoftheRNAwasdeterminedby
threemajorchemicalgroupsofAAS(ClarkandHenderson,2003).(1) measuringtheopticaldensityat260nm.FiftynanogramsoftotalRNA
Testosteroneesters,whicharederivedfromesterificationofthe17(cid:2)- wasthenreversetranscribedusingRETROscriptFirst-StrandSynthe-
hydroxylgroupoftestosterone,canbehydrolyzedintofreetestosterone, sis Kit for real time PCR (RT-PCR) (Ambion) in a total reaction
reducedto5(cid:1)-dihydrotestosterone(DHT)(Martini,1982;Winters,1990; volumeof20(cid:3)l.RNAwasdenaturedfor3minat75°Cwith2mM
KochakianandYesalis,2000)oraromatizedtoestrogens,including17(cid:2)- dNTPsand5(cid:3)Mrandomdecamers.TenunitsofRNaseinhibitor,RT
estradiol(Winters,1990;KochakianandYesalis,2000).Moleculesthat buffer to 1(cid:6), and 100 units of M-MLV reverse transcriptase were
havebeen5(cid:1)-reducedcannotbemetabolizedintoestrogens,butmaybe addedtothismixture,andthereactionwasincubatedfor1hat42°C,
metabolizedintootherandrogens,suchas3(cid:1)-androstanediol(3(cid:1)-diol). followedbyinactivationat92°Cfor10min.
(2)19-nor-testosteronederivatives.Thesecompoundshave,inconjunc- PCRprimersandTaqManMGBprobesspecificformouseGABA
A
tionwiththeadditionoflongsidechainmoieties,substitutionofa receptor(cid:1)subunitmRNAsweredesignedusingtheoligoprimerdesign
hydrogenforthemethylgroupatC19.Likethetestosteroneesters,19- programs Primer Express Software [Applied Biosystems (ABI)] and
nor-testosteronecanbearomatizedto17(cid:2)-estradiolandotherestrogens, OLIGO6(MolecularBiologyInsights)(Penattietal.,2005).Thespeci-
albeitwithlessefficiencythanfreetestosterone(Ryan,1959;Winters, ficityofeachprimer/probesetsequence,forindividualGABA receptor
A
1990). (3) C17-alkylated derivatives. The 17-methyl moiety precludes subunitswasconfirmedbyusingtheNationalCenterforBiotechnology
aromatization to 17(cid:2)-estradiol or reduction to DHT (Ryan, 1959; Information sequence alignment algorithm. Primers and probes for
QuinceyandGray,1967;Winters,1990),althoughproductionofother the (cid:2), (cid:4), (cid:5), and (cid:6) subunit mRNAs were obtained from ABI:
weak estrogens has been reported (Papaconstantinou et al., 2002; de Mm00433461_m1((cid:2)),Mm00549788_s1((cid:2)),Mm00433473-m1((cid:2)),
1 2 3
Gooyeretal.,2003). Mm00433476_m1 ((cid:5)), Mm00489932_m1 ((cid:6)), Mm00439047_m1 ((cid:4)),
1
EachanimalreceivedamixturecontainingequaldosesofoneAAS and Mm00433489_m1 ((cid:4)), as were primer/probes for ER(cid:1)
2
fromeachclass:2.5mg/kgtestosteronecypionate(TC),2.5mg/kgnan- (Mm00433149_m1), ER(cid:2) (Mm00599821_m1), aromatase (Cyp19)
drolonedecanoate(ND),and2.5mg/kg17(cid:1)-Methyltestosterone(17(cid:1)- (Mm00484049_m1), progesterone receptor A/B (Mm00435625_m1),
MeT)foratotalof7.5mgAAS/kg/dinsesameoil.Animalswereinjected KCC2 (Mm00803929_m1), GAD65 (Mm00484923_m1), and GAD67
for6of7dforaperiodof6weeks.ThistotaldailyAASconcentrationis (Mm00725661_s1). Amplification plots for designed primers corre-
comparable to high doses in human abusers (Kibble and Ross, 1987; spondingto(cid:1),(cid:1) and(cid:1) GABA receptorsubunitmRNAshadslopesof
1 2 5 A
PopeandKatz,1988;Perryetal.,1990).Controlsubjectswereadminis- (cid:2)3.36(for(cid:1) and(cid:1))and(cid:2)3.19(for(cid:1)),givingefficienciesof0.9844for
1 2 5
teredwiththesameinjectionparadigmandthesamevolume((cid:4)30(cid:3)l; (cid:1) and(cid:1) andof1.0581for(cid:1).
1 2 5
basedonbodyweight)withsesameoilalone.Foreachcohortstudied, RealtimePCRwasperformedusinganABI7500SequenceDetection
8–10 age-matched wild-type Tabby and AR-deficient Tfm littermates System.Validationexperimentswereconductedtoshowthatsubunit-
wereinjectedinparallel.Forelectrophysiologicalexperiments,tamox- specificprimersetsamplifiedwithequalefficiencyasreportedpreviously
ifen(2mg/kg/d)(RudickandWoolley,2003;MaguireandMody,2007), (Penattietal.,2005).EachmPOAsamplewasanalyzedintriplicatefor
formestane (4-hydroxyandrost-4-ene-3,17-dione or 4-OHA; 20 mg/ eachassessedcDNAandthe18SrRNAasaninternalstandard.Foreach
12486•J.Neurosci.,October7,2009•29(40):12484–12496 Penattietal.•AASEffectsintheForebrain
mRNAassessed,aPCRmastermixwasprepared,containingfinalcon- NP-40,1mMDTT,5%glycerol,1mMPMSF,10mMNaFplusaprotease
centrationsof:1(cid:6)TaqManUniversalMasterMix(containingAmpliTaq inhibitorcocktail,CompleteMini(Roche).Proteinconcentrationswere
Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, Passive determined using the BCA Protein Assay Reagent. Forty micrograms
Reference1,andoptimizedbuffercomponents),900nMforwardprimer, lysateforER(cid:1)wasseparatedby7.5%SDS-PAGEgelelectrophoresisand
900nMreverseprimer,250nMprobeinatotalreactionvolumeof25(cid:3)l. transferred to an Immobilon-P PVDF membrane (Millipore). Mem-
PCRfor18SrRNAwasperformedina25(cid:3)lofreactioncontaining1(cid:6) braneswereblockedin5%milk/TBST(10mMTris-HCl,pH8.0;150mM
TaqManUniversalMasterMix,1(cid:6)Eukaryotic18SrRNAEndogenous NaCl,0.2%Tween-20)for1hatroomtemperature.TheER(cid:1)antibody,
Control(VIC/MGBProbe,PrimerLimited;ABI),towhich1(cid:3)lofcDNA F-10 (s.c.-8002; Santa Cruz Biotechnology) was diluted 1:500 in 5%
wasadded.Thermocyclingconditionsincludedinitialstepsof2minat milk/TBST.Theanti-(cid:1)-tubulinantibody(DM1A,Calbiochem)wasdi-
50°C,10minat95°Cand40cyclesofPCRat95°Cfor15stodenature luted1:20,000in5%milk/TBS.Membraneswereincubatedovernightat
cDNAand60°Cfor1minforprimer/probeannealingandextension. 4°C,washedfivetimesfor5mineachinTBSTfollowedbyincubationfor
Samples with reverse transcriptase omitted were used to control for 1hwithagoatanti-mouseIgGsecondaryantibody(Pierce)at1:2000for
genomic DNA contamination and samples with template omitted to ER(cid:1)andfor(cid:1)-tubulinin5%milk/TBST.Antibodybindingwasdetected
controlforanyreagentcontamination.The2(cid:2)(cid:7)(cid:7)CTmethod(Livakand usinganenzyme-linkedchemiluminescencedetectionkit,SuperSignal
Schmittgen,2001;Peirsonetal.,2003)wasusedfordeterminationof WestFemtoMaximumSensitivityReagent(Pierce),andvisualizedon
mRNAlevels. autoradiographic film (Kodak). Densitometric measurements (Scion
Immunocytochemistry.Immunoreactivitywasassessedunderconfocal Image,Scion)oftheintensityofthesignalscorrespondingtoER(cid:1)and
microscopyasdescribedpreviously(Penattietal.,2005).Expressionof (cid:1)-tubulinwerecorrectedforbackgroundandtheER(cid:1)signalswerenor-
the(cid:1) subunitoftheGABA receptorwasassessedusingananti-guinea malizedtothatfor(cid:1)-tubulin.
2 A
pigpolyclonalprimaryantibody(1:15,000),generouslyprovidedbyDr. Electrophysiological recordings. Electrophysiological recording and
Jean-MarcFritschy(UniversityofZurich,Zurich,Switzerland)andvi- analyseswereperformedasdescribedpreviously(Yangetal.,2002;Pe-
sualized with Alexa Fluor 488 goat anti-guinea pig IgG (Invitrogen; nattietal.,2005;Jonesetal.,2006).Foreachexperiment,wholebrains
1:1000)(Penattietal.,2005).ImmunoreactivityforER(cid:1)wasassessed werequicklyremovedandplacedinice-coldoxygenatedlow-sodium
usingarabbitanti-ER(cid:1)antiserum(C1355;UpstateMilliporeBioscience sucrose-supplemented dissection solution, in mM: 250 sucrose, 1.2
ResearchReagents;1:1000)andvisualizedwithanti-goatAlexa594(1: CaCl ,10glucose,4KCl,7MgSO ,26NaHCO ,1.25NaH PO ,and1
2 4 3 2 4
1000dilution). ascorbicacidatpH7.35.UsinganElectronMicroscopySciencesOTS-
Steroidimmunoassays.Foreachassay,headsfromage-matchedTabby 4000Vibroslicer,250(cid:3)mcoronalsectionsthatincludedthemPOAwere
orTfmmalemicewererapidlyremovedandplacedinPBS(4°C;pH7.4) prepared.Slicesweresuperfusedwith95%O /5%CO -saturatedartifi-
2 2
towashawaytheexcessofblood.Brainswerequicklyremovedandthe cialCSF(aCSF),inmM:125NaCl,1.2CaCl2,10glucose,4KCl,1MgCl2,
mPOAs were dissected, separated from the underlying meninges and 26NaHCO ,1.25NaH PO ,and1ascorbicacidatpH7.35.
3 2 4
placedina1.5mlEppendorftube.Fortestosteronemeasurements,one Recordingsofspontaneousactionpotentials(APs)weremadeinthe
mPOA was assessed in duplicate from each control and AAS-treated on-cellconfigurationwithaCSFinboththebathandthepipetteatroom
animalofeachgenotype.Levelsof17(cid:2)-estradiolwerelowerthanthose temperature(20(cid:2)22°C).Datawererecordedtotapeandsubsequently
for testosterone and determinations were not reliable for assays from digitizedusingAcquire4.0software(BruxtonCorporation).Actionpo-
individual mPOAs. Therefore, determinations of 17(cid:2)-estradiol were tentialfrequencywasmeasuredandautocorrelogramswereconstructed
madefromsamplesforwhichmPOAtissuefromtwotofouranimalswas withbin-widthsof1–10msusingsoftwarewrittenlocallybyBrianL.
pooled.Thus,4–10animalspertreatmentconditionandpergenotype Jones(OregonHealth&ScienceUniversity,Portland,OR)inMatLab6.5
wereanalyzedforsteroiddeterminations.Eachtubewassupplemented R13(TheMathWorks),andrhythmicitywasassessedoverwindowsof
withPBS/5%ethanol;70(cid:3)lforeachtestosteronesampleor100(cid:3)lfor 0.2–10s.Largerbin-widthsandwindowswereusedforcellswithlower
each17(cid:2)-estradiolsample(pH7.4;roomtemperature).Tissuesamples firingrates.Classificationsoffiringpatternsfromautocorrelogramswere
werethenhand-homogenizedfor30sfollowedbycentrifugation(7000 madeaccordingtoBar-Gadetal.(2001).Categorizationofeachcell’s
rpm;15s).Thesupernatant,devoidofanycontaminatingtissue,was firingpatternasregular,irregular,orburstywasalsomadefromtheraw
thencarefullycollectedsoastonotdisturbthepelletoragitateandwas data. This independent assessment of firing pattern was in universal
storedovernightat(cid:2)20°C.Smallaliquots(6(cid:3)l)fromeachsupernatant agreementwithautocorrelogramdesignationsmadeindependentlyby
werecollectedforproteinassay(BCAProteinAssayReagent,Pierce). anotherobserver.
Supernatantsclearofanycontaminatingparticulatematterallowedthe ForrecordingsofGABA receptor-mediatedcurrents,aCSFwassup-
A
resultingsamplestobeassayeddirectlywithExpandedRangeTestoster- plementedwith2mMkynurenicacidtoblockexcitatorytransmission,
oneandHighSensitivityEstradiolSalivaryImmunoassayKitsfromSali- andrecordingsweremadeinaCSFatroomtemperature(20(cid:2)22°C)in
metrics.ThesetwoELISAkitshavelowdetectionthresholdsandsmall thewhole-cellconfigurationofthepatch-clamptechniqueataholding
volumerequirements(6pg/ml;25(cid:3)land1pg/ml;100(cid:3)lfortestosterone potentialof(cid:2)70mV.Theinternalelectrodesolutionconsistedof(in
and 17(cid:2)-estradiol, respectively). Optical densities were determined at mM):153CsCl,1MgCl2,5EGTA,and10HEPES,towhich2MgATPwas
450nmaccordingtothekit’sinstructionsandsteroidconcentrations added for every experiment. GABA receptor-mediated spontaneous
A
wereconvertedfrompg/mltopg/mgmPOAtissue.Foreachassay,re- IPSCs(sIPSCs)wereisolatedfromaminoacid-mediatedexcitatorysyn-
coveryrates((cid:4)60%)weredeterminedbysupplementationoffiveknown apses by addition of 2 mM kynurenic acid to the bath and miniature
concentrationsofeachsteroidtosamplescontainingmPOAhomoge- IPSCs(mIPSCs)isolatedbyadditionalperfusionwith1(cid:3)Mtetrodotoxin
natesofequivalentproteinconcentrationandrecoveryforeachknown (TTX)(Mozrzymasetal.,1999).ToconfirmthatIPSCsweremediated
concentrationwasdeterminedinduplicate.Therecoveryratesforeach byGABAAreceptors,10(cid:3)Mbicucullinewasused,insomeexperiments,
ofthefiveknownconcentrationswereaveragedtoprovideanaverage toreversiblyblocktheevents.Foranalysisofpeakcurrent(I )and
peak
recoveryrateforeachsteroid,andrawdeterminationsoftheconcentra- currentdecays(biphasicandfittedwithtwotimeconstants,(cid:7) and(cid:7);
1 2
tions of testosterone and 17(cid:2)-estradiol were corrected for recovery. Nettetal.,1999;Penattietal.,2005)sIPSCsormIPSCswereacquiredand
Analysisofintra-andinter-assayvariabilitywereestimatedfrom10de- averaged from each neuron for a period of 4–12 min per recording
terminationsoftwosetsofthreesamplesofmPOAhomogenatescon- condition, with criteria imposed that seal resistance ((cid:1)1 G(cid:9)), access
taining5concentrationsofeachsteroidasthe%CV(CV*100)forthe resistance((cid:8)25M(cid:9))andholdingcurrentdidnotchangemorethan
highestvariationobtained(Steineretal.,2003;vanGeeletal.,2008):for (cid:10)(cid:4)10%duringtherecording.Eventswereacceptedforpeakcurrent
testosterone: %CV(intra-assay): (cid:8)0.43% and %CV(inter-assay): only if they had rise-times of (cid:8)2.5 ms and were isolated from other
(cid:8)1.5%;for17(cid:2)-estradiol:%CV(intra-assay):(cid:8)1.0%and%CV(inter- synapticevents(i.e.,asynchronous,butconcurrentreleasedidnotob-
assay):1.8%. scureeithertherisingorthedecayphaseofthemeasuredresponse).For
Westernblotanalysis.Forproteinextraction,mPOAtissuewasho- analysisofcurrentamplitudesanddecaykineticsforwhichoverlapping
mogenizedin25mMTris-HCl,pH7.5,150mMNaCl,5mMMgCl2,1% eventswerenotmeasured,thetotalnumberofeventsanalyzedpercell
Penattietal.•AASEffectsintheForebrain J.Neurosci.,October7,2009•29(40):12484–12496•12487
rangedfrom(cid:1)180to(cid:8)30foraminorityofcellsinwhichallcriteriawere
metwithrespecttoqualityofrecording.Coefficientsofvariationdidnot
varywiththenumberofeventsanalyzed(e.g.,therangeforvaluesofI
peak
was0.34–0.53).Alleventsthatexceededthresholdfordetectionwere
acceptedforassessmentofIPSCfrequencies.Datawererecordedtotape
andanalyzedusingMiniAnalysis(Synaptosoft).Inadditiontoestimates
of(cid:7) and(cid:7),overallsynapticcurrentdecaywasalsodescribedbyasingle
1 2
weightedtimeconstant((cid:7) )(Yangetal.,2002,2005;Jonesetal.,2006).
w
ThemagnitudeoftonicGABA receptor-mediatedcurrents(I )at-
A tonic
tributedto(cid:1)-containingreceptorswereestimatedbyminormodifica-
5
tions of previously published procedures (Farrant and Nusser, 2005;
Jonesetal.,2006)asachangeinthebaselineholdingcurrent(I )in
baseline
thepresenceof50(cid:3)M(0.1%DMSO)ofthe(cid:1)5-selectiveinverseagonist,
L-655,708(Quirketal.,1996);aconcentrationthathasbeenreportedto
beselectivefor(cid:1)-containingreceptorsinslicepreparations(Caraiscos
5
etal.,2004;Scimemietal.,2005)andshowntobewithouteffectin(cid:1)(cid:2)/(cid:2)
5
mice (Caraiscos et al., 2004). Acquisition of data in the presence of
L-655,708wasinitiated(cid:4)1minafterthedrugwasperfusedintothebath,
andacquireddatawereanalyzedforaperiodof90–150sfollowedbya
washperiod.Allcontrolrecordingswereperformedin0.1%DMSOfor
comparison. This concentration of vehicle had no effect on GABA
A
receptor-mediated responses (i.e., no significant differences between
aCSF alone and aCSF/0.1% DMSO), as we have reported previously
(Jonesetal.,2006).
Drugs.AllchemicalswerepurchasedfromSigmaChemicalwiththe
exceptionofL-655,708(TocrisBioscience).
Statisticalanalyses.Valuesforvariablesarepresentedasmean(cid:10)SE.
Shapiro–WilksorKolmogorov–Smirnovtestswereusedtodetermineif
data were distributed normally. For electrophysiological experiments,
fornon-normallydistributeddata,valueswerelog-transformedbefore
significancebeingdeterminedbyone-ortwo-wayANOVAusingthe
generallinearmodelprocedureofSASfollowedbytheStudent’sttestor
meanscomparisonbyleastsignificantmeans,respectively.Significance
forautocorrelationalanalysisofactionpotentialfiringwasassessedby
theX2test.Forreal-timePCRanalysis,CTvaluesweredefinedasoutliers
when (cid:10)3 SDs from the mean. Results were qualitatively the same
whetherornotoutlierswereincludedinthefinalanalysis.Differencesin
therelativeabundanceofeachmRNAforAAS-treatedversuscontrol
subjectswereassessedusingPair-WiseFixedReallocationRandomiza-
tion t test using the excel-based Relative Expression Software Tool
(REST)(Pfaffl,2001;Pfaffletal.,2002).FormRNAanalysis,onlypositive
errorbarsaredepictedintheresults;positiveandnegativeerrorbars
differedby(cid:8)15%.Foralldata,the(cid:1)levelwassetatp(cid:8)0.05.Through-
outthetext,nvaluesindicatethenumberofanimalsanalyzedpercon-
Figure1. EffectsofchronicAAStreatmentonAPfrequencyandpatterninginwild-typeand
dition, except for electrophysiological analyses where n indicates the
AR-deficientmalemice.A,AverageAPfrequencyundercontrol(black)andAAStreatment
numbers of cells analyzed per condition. The probability of attaining
(gray)conditionsfromwild-type(left)andAR-deficient(right)mice.Asterisksindicatevalues
recordingsthatmetcriteriaforsubsequentanalyses(1–3cellspersubject
fromAAS-treatedsubjectsweresignificantlydifferentfromtheirage-matchedoil-injectedcon-
forbothcontrolandtreatedgroups)islowintheseolderanimals((cid:1)100
trolsinthesamegenotype.B,Representativeexamplesofactionpotentials(top)andautocor-
dofageatthetimeofrecording),andnoonesubjectwithinagivengroup
relograms(bottom)correspondingtoirregular,bursty,andregularfiringpatterns.C,The
disproportionatelycontributedcellstothegroupaverages.
percentageofneuronsdisplayingirregular,burstyorregularfiringpatternsasassessedby
autocorrelationalanalysesofMPNneuronsfromcontrolorAAS-treatedwild-type(left)orAR-
Results deficient(right)mice.Recordingsweremadefrom8miceforeachtreatmentconditionand
eachgenotype.
I.AAS-inducedchangesinelectricalactivityinMPNneurons
inwild-typemalemice
Electrophysiological recordings were made from the dorsal as- cientofvariationinAPfrequency(p(cid:5)0.0040)inMPNneurons
pectoftheMPN,whichiscentrallysituatedwithinthemPOA. fromAAS-treatedversuscontrolwild-typemice.
ChronictreatmentwiththeAASmixtureresultedinasignificant
(p(cid:5)0.0010)increaseinAPfrequencyfrom1.83(cid:10)0.33inMPN II.AAS-inducedchangesinGABA receptorexpressionand
A
neuronsfromcontrolwild-typemice(n(cid:5)32neurons)to4.31(cid:10) functioninwild-typemice
0.74 (n (cid:5) 30 neurons) in AAS-treated subjects (Fig. 1A). This ChangesinGABA receptorsubunitmRNAlevelswereassessed
A
increasewasreflectedacrosstheentirerangeofAPfrequencies forthemajorsubunitisoformsofthereceptorexpressedinthe
observed. Autocorrelational analysis indicated that firing pat- mPOA((cid:1),(cid:1),(cid:1),(cid:2),(cid:2),(cid:2),(cid:4),(cid:4),and(cid:6))(Henderson,2007),as
1 2 5 1 2 3 1 2
ternsofMPNneuronscouldbedescribedasirregular,burstyand wellasthe(cid:5)subunitwhich,whileonlyweaklyexpressedinthe
regular based on interspike interval (Fig. 1B). AAS treatment mPOA, may contribute to extrasynaptic tonic conductances in
resultedinatrendtowardadecreaseinneuronswithburstyfiring this brain region, as it does in others (Semyanov et al., 2004).
(p(cid:5)0.0550)(Fig.1C)andasignificantdecreaseinthecoeffi- Real-timePCRanalysisrevealedamarkedincreasein(cid:1) subunit
5
12488•J.Neurosci.,October7,2009•29(40):12484–12496 Penattietal.•AASEffectsintheForebrain
mRNA with AAS treatment in wild-type
mice (p (cid:5) 0.0010; n (cid:5) 8) (Fig. 2A). A
small, but nonetheless significant, in-
creasein(cid:2) subunitmRNA(p(cid:5)0.0300;
1
n(cid:5)8)wasalsoobservedinAAS-treated
wild-typesubjectsrelativetothissubunit
mRNAlevelsobservedincontrolanimals.
While the level of (cid:1) mRNA was in-
5
creased,weobservednoconcomitantin-
creaseinthelevelsofeither(cid:5)or(cid:6)subunit
mRNAsinthemPOAofAAS-treatedan-
imals (Fig. 2A); subunits which would
likelybepresentinanynewextrasynaptic
receptors whose expression was induced
byAAStreatment(Henderson,2007).
Receptors containing the (cid:1) subunit
5
contributetobothsynapticandextrasyn-
aptic (tonic) currents in other brain re-
gions(Dunningetal.,1999;Caraiscoset
al.,2004;Serwanskietal.,2006;Yamadaet
al.,2007;AliandThomson,2008).Tode-
terminehowtheAAS-dependentincrease Figure2. EffectsofchronicAAStreatmentonGABA receptorexpressionandfunctioninthemPOAofwild-typemalemice.A,
A
in (cid:1) subunit mRNA in wild-type mice DataindicatingaveragelevelsofsubunitmRNAsintissueisolatedfromthemPOAfrom8wild-typemicetreatedwiththeAAS
5
maybereflectedinchangesinGABAergic mixturefor6weeks.ThemRNAlevelsforeachsubunitnormalizedto18SrRNAfrom8oil-injectedcontrolanimalswereanalyzed
transmission, GABA receptor-mediated andrelativesubunitmRNAlevelsforcontrolsweresetto1.00foreachsubunitmRNA(horizontalline).Values(2(cid:2)(cid:7)(cid:7)CT)for
A treatedanimalswereplottedrelativetothiscontrolvalueof1.00.B,AveragedsIPSCsrecordedfromMPNneuronsinslicesisolated
synapticandtoniccurrentswererecorded
fromcontrol(blackline)andAAS-treated(grayline)wild-typemice.C,D,AverageI (left),(cid:7) (center)andchargetransfer(Q ;
fromMPNneuronsofcontrolandAAS- peak w tot
right)(C)andfrequencyforsIPSCsfromtheseMPNneuronsofcontrol(blackbars)andAAS-treated(graybars)mice(D).Asterisks
treatedwild-typeanimals.
indicatevaluesfromAAS-treatedsubjectsthatweresignificantlydifferentfromtheirage-matchedoil-injectedcontrols.Record-
In control wild-type mice, sIPSCs in
ingsweremadefrom16miceforeachtreatmentcondition.
MPN neurons were characterized by a
mean peak current amplitude of 40.7 (cid:10) al.,1999;SmithandGong,2005).Acuteapplicationofthe(cid:1)-
3.8pA(Fig.2B,C),biexponentialcurrentdecaydescribedbytwo 5
selective antagonist, L655,708, to MPN neurons suggested that
timeconstantsof9.96(cid:10)0.49and41.00(cid:10)4.11ms(aweighted (cid:1)-containing receptors contribute to synaptic responses in
timeconstant(cid:7)w(cid:5)21.29(cid:10)1.63ms)(Fig.2B,C),andamean (cid:4)550% of MPN neurons tested from both control and AAS-
frequencyof4.7(cid:10)0.8Hz(Fig.2D)(n(cid:5)22cells).AAStreatmentof
treatedmice.Forthoseneuronsthatweresensitivetotheeffects
wild-type mice did not significantly increase sIPSC amplitude or ofL655,708,(cid:7) wassignificantly(p(cid:5)0.0072)longerinneurons
frequency(Fig.2B,D),butdidresultinanoverallslowingofsynap- fromAAS-treawtedthanfromcontrolmice(23.07(cid:10)1.66msfor
ticcurrentdecay((cid:7)w;p(cid:5)0.027)(Fig.2C),arisingfromanin- control,n(cid:5)5neurons,versus32.37(cid:10)1.84msforAAS-treated,
creasein(cid:7)1(p(cid:5)0.0440)andatrendtowardprolongationof(cid:7)2in n(cid:5)5neurons),consistentwiththeinterpretationthatthereis
AAS-treatedversuscontrolwild-typemice.Theoverallslowing increasedrepresentationof(cid:1)-containingreceptorsinthepop-
ofsynapticcurrentdecayalsocontributedtoasignificant(p(cid:5) 5
ulationofsynapticreceptorsofAAS-treatedwild-typemice.An
0.0450)increaseintotalchargetransfer(Qtot)intheAAS-treated L655,708-sensitive tonic current (I (cid:5) 7.7 (cid:10) 4.3 pA; n (cid:5) 8
versuscontrolsubjects(Fig.2C). tonic
neurons)wasalsoevidentinMPNneuronsfromwild-typemice,
Asnotedabove,AAStreatmentincreasedAPfiringfrequencyin
butwhileAAStreatmentaugmentedtheamplitudeofthiscur-
MPNneuronsof wild-type mice. An AAS-dependent enhance- rent(16.0(cid:10)13.1pA;n(cid:5)10neurons),thisdifferencewasnot
ment of GABAergic transmission could directly give rise to an
significant.
increaseinAPfiringfrequencyifGABAactstodepolarizeneu-
ChronicexposuretothismixtureofcommonlyabusedAAS
ronswithinthemPOA.Ithasbeenshownthatperturbationsin
resultedinsignificantdifferencesinAPfrequencyandpattern-
normalneuronalsignalingthatpromotestresscancauserever-
ing, GABA receptor subunit mRNA expression and GABA
siontodepolarizingactionsofGABAintheadulthypothalamus A A
receptor-mediated synaptic responses in wild-type mice. As
byalteringtheexpressionoftheaniontransporter,K(cid:3)/Cl–co-
notedpreviously,theAASinthismixturenotonlyinteractwith
transporter2(KCC2)(Hewittetal.,2009).WhileAAStreatment
AR,butalsohavethecapacitytobearomatizedtoestrogensand
isknowntobeassociatedwithchangesinthestressresponse(for
topotentiallyinteractwithER.TodeterminetheroleoftheARin
review,seeClarkandHenderson,2003),real-timePCRanalysisof
mediating the effects of this AAS mixture on neuronal activity
mPOAtissueindicatedthatAAStreatmentdidnotresultinany
andGABA receptorexpressionandfunctioninthemPOA,re-
significantchangeinKCC2mRNAexpressioninthisbrainre- A
cordings were next made from control and AAS-treated AR-
gion(p(cid:5)0.3245).
deficientTfmmice.
TheslowingofsIPSCcurrentdecayandtheincreaseintotal
chargetransferobservedinAAS-treatedwild-typemicewouldbe III.AAS-inducedchangesinelectricalactivityinMPN
consistentwithanincreaseintheincorporationof(cid:1) subunits neuronsinAR-deficient(Tfm)malemice
5
intosynapticreceptors,sinceinclusionofthissubunithasbeen Incontrasttoitseffectinwild-typeanimals,AAStreatmentofthe
reportedtoconfermoreslowlydecayingresponsesrelativetorecep- AR-deficient mice resulted in a significant decrease in AP fre-
torscontainingother(cid:1)subunits(Burgardetal.,1996;Dunninget quency(p(cid:5)0.0240),from3.04(cid:10)0.52inMPNneuronsfrom
Penattietal.•AASEffectsintheForebrain J.Neurosci.,October7,2009•29(40):12484–12496•12489
AAS treatment, the average I in MPN neurons from AAS-
peak
treated mice (n (cid:5) 28 neurons) was significantly (p (cid:5) 0.0034)
reducedto71%ofthatinMPNneurons(n(cid:5)25neurons)from
controlAR-deficientsubjects(Fig.4A,B).ThesmallersIPSCam-
plitudes in AAS-treated subjects may arise from postsynaptic
changes, either a decrease in postsynaptic receptor density or
decreasedchargetransferthroughthesereceptors,ormayarise
fromdiminishedAP-dependentpresynapticrelease.Spontane-
ous IPSC frequency for MPN neurons from AAS-treated AR-
deficientmicewassignificantly(p(cid:5)0.0191)lower(47%)than
thatforneuronsfromcontrolanimals(4.77(cid:10)0.76Hz;n(cid:5)29
neuronsfromAAS-treatedvs10.04(cid:10)2.18Hz;n(cid:5)25neurons
forcontrol)(Fig.4C).NodifferenceswereobservedinmIPSC
amplitude,decaykinetics,totalchargetransferorfrequency(Fig.
4D–F)incontrolversusAAS-treatedAR-deficientmice(n(cid:5)23
neurons from control and n (cid:5) 22 neurons from AAS-treated
mice), suggesting that neither the decrease in I nor the de-
peak
crease in sIPSC frequency in AAS-treated subjects was due to
diminished postynaptic sensitivity, which, for analysis of fre-
quencymayhaveresultedinanapparent“loss”ofsmalleventsin
thebaselinenoise.Rather,theAAS-dependentdecreaseinI
peak
andfrequencyofsIPSCsintheMPNobservedintheAR-deficient
mice(Fig.4A–C)isconsistentwithdecreasedAPfiringfrequency
observedinMPNneuronsofAAS-treatedAR-deficientmiceand
concomitantAP-dependentpresynapticrelease.
Figure3. GABA receptorsubunitmRNAlevelsinthemPOAofwild-typeandAR-deficient
A AsafurtherassessmentofAASeffectsonGABAergictone
mice.A,Dataarepresentedasthe2(cid:2)(cid:7)CTvaluesindicatingtherelativelevelsofGABA receptor
A intheMPN,wealsodeterminedtheeffectsofchronicsteroid
subunitexpressioninthemPOAofcontrolwild-type(black)andcontrolAR-deficient(white)
treatment on the levels of the mRNAs encoding the two iso-
mice.B,DataindicatingaveragelevelsofsubunitmRNAsintissueisolatedfromthemPOAfrom
AR-deficientmicetreatedwiththeAASmixturefor6weeks.FordatashowninB,valuesfrom forms of glutamic acid decarboxylase (GAD), the synthetic
oil-injectedcontrolanimalswereanalyzedandrelativesubunitmRNAlevelsforcontrolswere enzymeforGABA.The65kDaisoformofthisenzyme,GAD ,
65
setto1.00foreachsubunitmRNA(horizontalline).The2(cid:2)(cid:7)(cid:7)CTvaluesforAAS-treatedani- is preferentially localized in axon terminals, and levels of
malswereplottedrelativetothiscontrolvalueof1.00.Analysesweremadefrom8controland GAD immunoreactivity strongly correlate with levels of
65
8treatedanimalsofeachgenotype. GAD mRNA (Esclapez et al., 1994). GAD is strongly ex-
65 65
pressed in the mPOA, and levels of this transcript vary in
controlmice(n(cid:5)31neurons)to1.72(cid:10)0.37inMPNneurons parallelwiththechangesinphasicactivityinneuroendocrine
fromAAS-treatedmice(n(cid:5)30neurons)(Fig.1A),andthis cellsinthisandotherhypothalamicregions(Feldblumetal.,
decrease was observed across the range of observed frequen- 1993).AAStreatmentofAR-deficientmiceledtoasignificant
cies.Incontrasttoitseffectsinwild-typemice,AAStreatment (p (cid:5) 0.0425) decrease in the levels of GAD mRNA in the
65
didnotappreciablyalterthepatterning(Fig.1C)orthecoef- mPOA to 75% that observed in control animals. AAS treat-
ficient of variation for AP firing in the MPN of these AR- mentdidnotalterthelevelsofGAD mRNA.Thesedataare
67
deficientmice. alsoconsistentwithdiminishedpresynapticGABAergictone
Theprofileofsteady-statelevelsofGABA receptorsubunit intheMPNofAAS-treatedTfmmice.AAStreatmentdidnot
A
mRNAsinthemPOAofcontrolAR-deficientTfmmicewascom- significantlyalterGAD (orGAD )levelsinwild-typemice,
65 67
parabletothatobservedinwild-typeTabbymalemice.Ofpar- consistent with an absence of effect of AAS-treatment on
ticularnote,levelsofthe(cid:1) subunitmRNAwerenearlyidentical sIPSCamplitudeorfrequencyinwild-typesubjects.
5
(Fig.3A).Incontrasttotheincreasesin(cid:1) and(cid:2) subunitmRNAs
5 1
observedinthemPOAwithAAStreatmentofwild-typemice, IV.EffectsofERantagonismonAAS-dependentchangesin
AAStreatmentofAR-deficientmicedidnotresultinincreases GABAergicsignalingintheTfmmouse
(compared with control) in any of the examined GABA re- ThesignificanteffectsofchronicAAStreatmentinthemPOAof
A
ceptorsubunitmRNAsinthisbrainregion.Ofparticularin- Tfmmicesuggestthat,intheabsenceoffunctionalARsignaling,
terest, the marked increase in (cid:1) subunit mRNA that was significant interactions of the AAS with ER signaling pathways
5
observed in wild-type mice was notably absent in this AR- wereunmasked.WhileAASsuchas17(cid:1)-MeTdonotbindtoER
deficient mutant line. Rather, real-time PCR analysis revealed (Oosterkampetal.,1996),allAASusedinthismixturecanbe
modest,albeitsignificant,decreasesin(cid:2) (p(cid:5)0.0200)and(cid:4) (p(cid:5) metabolizedtoestrogens(videsupra)andthushavethepropen-
3 2
0.0060)subunitmRNAsinAAS-treatedversuscontrolAR-deficient sitytoactasERagonists.Inaddition,innon-neuronalcellsas-
mice(Fig.3B). sessed in vitro, the AAS have been shown to inhibit aromatase
FortheAR-deficientTfmmice,consistentwiththelackofan (Moretal.,2001;deGooyeretal.,2003)andthuscouldindirectly
AAS-dependentincreasein(cid:1) mRNA,AAStreatmentpromoted acttoimpairERsignalingbylimitingtheavailabilityofestrogens
5
neitheraprolongationofsynapticcurrentdecaynoranincrease inthebrain.Experimentswerenextperformedtodetermineif
intotalchargetransferintheseanimals(Fig.4A,B);changesin the ability of the AAS to modulate GABAergic signaling in the
currentparametersthataccompaniedtheincreasein(cid:1) subunit MPNintheAR-deficientTfmmousecouldbeattributedtothese
5
mRNA in the mPOA of wild-type males. To the contrary, al- compoundsactingbyeitherofthesetwomechanismstoaugment
thoughtherewasnochangeinsIPSCdecaykineticsinducedby ordiminish(respectively)ER-mediatedsignalinginthemPOA.
12490•J.Neurosci.,October7,2009•29(40):12484–12496 Penattietal.•AASEffectsintheForebrain
TofirstdetermineifAASeffectsinthe
AR-deficientmicecouldbeattributedto
agonisticactionsattheER,Tfmmicewere
treatedwiththeselectiveestrogenrecep-
tormodulator(SERM),tamoxifen,orthe
AASmixtureinconjunctionwithtamox-
ifen.Tamoxifenatthedosegivenherehas
been shown to be devoid of acute estro-
genic activity, but to possess ER antago-
nisticactivityintherodentbrain(Rudick
andWoolley,2003).Surprisingly,concur-
renttreatmentofAR-deficientmicewith
tamoxifendidnotantagonizetheactions
of AAS treatment on GABAergic trans-
mission in the MPN of the AR-deficient
mice,aswouldhavebeenexpectedifthe
AASwereactingasestrogensandtamox-
ifen as an ER antagonist. Instead, we
foundthattamoxifenalonealsodecreased Figure4. EffectsofchronicAAStreatmentonGABA receptorfunctionintheMPNofAR-deficientmice.A,AveragedsIPSCs
A
sIPSCamplitudesversuscontrolsubjects recordedfromMPNneuronsinslicesisolatedfromcontrolandAAS-treatedAR-deficientmice.B,C,AverageI (left),(cid:7) (center)
peak w
(p(cid:8)0.0001)(Fig.5)andthattherewas andchargetransfer(Q ;right)(B)andfrequencyforsIPSCsfromtheseMPNneuronsofcontrolandAAS-treatedmice(C).D,
tot
no difference in Ipeak between AR- AveragemIPSCsrecordedfromMPNneuronsofcontrolandAAS-treatedAR-deficientmice.E,F,AverageIpeak(left),(cid:7)w(center),
deficient animals treated concurrently andchargetransfer(Qtot;right)(E)andfrequencyformIPSCsfromtheseMPNneurons(F).Blacklinesandbars,Control;graylines
andbars,AAS-treated.AsterisksindicatevaluesfromAAS-treatedsubjectsthatweresignificantlydifferentfromtheirage-
withtheAASplustamoxifenversusthose
matchedoil-injectedcontrols.Recordingsweremadefrom12to16miceforeachtreatmentcondition.
treatedwithtamoxifenalone(Fig.5).As
withAAStreatment,tamoxifenalsopro-
Gooyeretal.,2003)versustheirreversibleinhibitionofthisen-
motedasignificantdecreaseinthefrequencyofsIPSCsinMPN
zymeimposedbyformestane(YueandBrodie,1997;Kohleret
neurons of AR-deficient Tfm mice treated with tamoxifen versus
controlsubjects(3.60(cid:10)0.74Hzvs10.04(cid:10)2.18Hz;p(cid:5)0.0306). al., 2007). As with AAS-treatment, the frequency of sIPSCs in
Therewasnodifference(p(cid:5)1.0000)forsIPSCfrequenciesbetween MPNneuronsofanimalstreatedwithformestanewasalsolower
than in control subjects (5.67 (cid:10) 0.92 Hz vs 10.04 (cid:10) 2.18 Hz),
AAS-andtamoxifen-treatedTfmmice.Whilerecognizingthatta-
althoughforformestanealone,thisdifferencedidnotattainsig-
moxifenisaSERMwithmixedagonistic/antagonisticproperties(see
nificance. There was no difference in frequency for sIPSCs in
for example, Shang et al., 2000; Kressler et al., 2007), these data
MPN neurons from AAS-treated and formestane-treated AR-
suggestthattheAASdonotactasERagonistsintheAR-deficient
deficientTfmmice.Together,thesedataareconsistentwiththe
mice.Rather,thedatasuggestthatthemaybeinterferingwiththe
hypothesisthat,intheabsenceofARsignalingintheTfmmouse,
actionsofestrogensattheERormaybeactingtolimittheirproduc-
theAASdiminishGABAergicsignalingbylimitingtheproduc-
tionviainhibitionofaromatase.
tionofestrogensandthusER-mediatedsignaling.
IfintheabsenceoffunctionalAR,theabilityoftheAASto
V.EffectsofaromataseinhibitorsandestrogenonAAS-
dependentchangesinGABAergicsignalingintheTfmmouse diminish GABAergic transmission reflects inhibition of aro-
Todirectlydeterminewhetherinhibitionofendogenousestro- matase, and thus production of estrogen, supplementing AAS-
genproductioncouldmimictheeffectsoftheAASonGABAergic treated Tfm mice with exogenous estrogen should restore the
transmissioninAR-deficientmice,Tfmmalesweretreatedwith properties of GABAA receptor-mediated transmission to those
formestaneorwiththeAASmixtureinconjunctionwithform- observed in control mice. To this end, an additional cohort of
estane.Formestaneisaselectiveandirreversiblearomatasein- AR-deficientmicewastreatedwithoilalone,theAASmixturein
hibitor (Yue and Brodie, 1997; Kohler et al., 2007), which will conjunctionwith17(cid:2)-estradiolor17(cid:2)-estradiolalone.Consis-
blockproductionofestrogensfromandrogensincludingthose tent with the hypothesis that the AAS are acting as aromatase
AASthatcannotbe5(cid:1)-reduced(LaMorteetal.,1994).Formestane inhibitorsintheTfmanimals,averagesIPSCamplitudeswerenot
hasbeenshowntocrossthebloodbrainbarrierandtodiminish significantly different between control animals and animals
aromataseactivityandER-regulatedproteinexpressioninthebrain treated with AAS in conjunction with a concentration of 17(cid:2)-
(Foidartetal.,1994;deFougerollesNunnetal.,1999). estradiolintendedtorestorelevelsofthissteroidtoaphysiolog-
AsinAR-deficientTfmmicetreatedwithAAS,sIPSCampli- ical range (Fig. 5). While average I in animals treated with
peak
tudesinMPNneuronsofTfmmicetreatedwithformestanewere 17(cid:2)-estradiol alone was greater than for control subjects, the
significantlylower(p(cid:8)0.0001)thanthoseobservedincontrol differencedidnotattainsignificance(Fig.5).Asreportedabove
subjects (Fig. 5). Moreover, there was no difference in sIPSC (Section III), AAS treatment of Tfm mice significantly (p (cid:5)
amplitudesbetweenmicetreatedwiththeAASandthoseconcur- 0.0191)decreasedsIPSCfrequencyto47%thatobservedincon-
rentlytreatedwithformestaneandtheAAS(Fig.5).Thediminu- trol mice, but concurrent treatment of the AR-deficient mice
tionofsIPSCpeakamplitudeswas,however,significantly(p(cid:5) withAASand17(cid:2)-estradiolrestoredtheaveragesIPSCfrequency
0.0005) greater in animals treated with formestane alone than to95%thatobservedforcontrols(p(cid:5)0.8848).Treatmentof
thosetreatedwiththeAAS(Fig.5).Theseresultsmayreflectthe AR-deficientmicewith17(cid:2)-estradiolaloneevokedanearlysig-
competitiveandincompleteinhibitionofaromataseactivityby nificant(p(cid:5)0.0539)increaseinsIPSCfrequency(175%con-
theAAS(invitroassaysindicate17(cid:1)-MeTandnandrolonein- trol). As with AAS mixture treatment of Tfm animals, no
hibit aromatase activity by 53% and 85%, respectively) (de significantdifferenceswereelicitedinsynapticcurrentdecayby
Penattietal.•AASEffectsintheForebrain J.Neurosci.,October7,2009•29(40):12484–12496•12491
Table1.Levelsoftestosteroneand17(cid:1)-estradiolinmPOAtissue
Genotype/treatment T(pg/mgprotein) E (pg/mgprotein) Aromatization(E /T)
2 2
Wildtype(control) 18.54(cid:10)9.35 1.36(cid:10)0.21 0.074
Wildtype(AAS) 153.30(cid:10)19.60 2.11(cid:10)0.51 0.014
AR-deficient(control) 4.96a 0.78(cid:10)0.08 0.157
AR-deficient(AAS) 135.65(cid:10)20.4 0.33(cid:10)0.04 0.002
Mean(cid:10)SEofthemeanlevelsoftestosterone(T)and17(cid:2)-estradiol(E )inmPOAtissueharvestedfromwild-type
2
control(n(cid:5)4miceforT;n(cid:5)8forE )orAAS-injected(n(cid:5)4miceforT;n(cid:5)10miceforE )orAR-deficientcontrol
2 2
(n(cid:5)6miceforT;n(cid:5)8miceforE ),orAAS-injected(n(cid:5)4miceforT;n(cid:5)8miceforE )malemice,andthe
2 2
respectiveratioofaromatizationasdeterminedfromtheratioofE toT.
2
aLevelsoftestosteronewereabovethresholdfordetectioninonly1of6mPOAsamplesfromAR-deficientanimals.
Fortheother5animals,thethresholdfordetection(3.0pg/mg)wasenteredfordeterminationofthemean,butno
SEwascalculated.AsnotedinMaterialsandMethods,individualmPOAwereassayedfortestosterone.Asindicated
inMaterialsandMethods,for17(cid:2)-estradiol,determinationsweremadefromsamplesforwhichmPOAtissuefrom
2to4animalswerepooled.
derivedfromtestosteroneiscapableofsuppressingfolliclestim-
ulatinghormonesecretionintheTfmmouse,thusindicatinga
physiological ER-mediated response of the hypothalamic/pitu-
itary/gonadal axis in this mutant mouse line (Schleicher et al.,
Figure5. EffectsofpharmacologicalmanipulationofestrogensonAAS-dependentmodulation
ofGABAergicsIPSCamplitudesintheMPNoftheAR-deficientTfmmouse.Graphicalrepresentationof 1989).
averagedI fromAR-deficientTfmmicechronicallytreatedwithoilalone(Control),theAASmixture Todetermineifaromatizationoftestosteronetoestrogenin
peak
inoil(AAS),tamoxifen(Tx),theAASmixtureandtamoxifen(Tx(cid:3)AAS),formestane(Frm),ortheAAS themPOAwasalteredbyAAStreatment,steroidimmunoassays
mixtureandformestane(Frm(cid:3)AAS).DataforControlandAASarethesameasshowninFigure5. were performed to assess levels of these steroids in this brain
Identicalletters(amongthea–ddesignations)indicatemeansthatwerenotstatisticallydifferent region.TestosteronelevelswerenotablyhigherformPOAtissue
fromoneanotherasassessedbytwo-wayANOVAfollowedbythemeanscomparisonbyleastsignif- frommalecontrolwild-typethanfromcontrolAR-deficientan-
icantmeans.InaseparatecohortofAR-deficientTfmmice,experimentswerelaterperformedto
imals(Table1).Thesedataareconsistentwithpreviousstudies
determinetheeffectsofconcurrenttreatmentwith17(cid:2)-estradiolandtheAASmixture(E(cid:3)AAS)or
2 assessing levels of peripheral testosterone in adult male mice
17(cid:2)-estradiol(E)aloneversuscontrol.Theidenticalletter(a’)indicatesthatwhenconcurrently
treatedwith17(cid:2)2-estradiolandAAS,theAASdidnotsignificantlydiminishI .Forbothsetsofdata, (Murphy and O’Shaughnessy, 1991; O’Shaughnessy and Mur-
peak phy, 1993; Vandenput et al., 2004). As expected, we found
numbersinparenthesesindicatenumbersofcells.Recordingsweremadefrom8to12miceforeach
treatmentcondition. marked elevation of testosterone within the mPOA of AAS-
treatedversuscontrolmiceinbothwild-type(p(cid:5)0.0008)and
AR-deficient(p(cid:5)0.00004)animals(Table1),reflectingthesig-
concurrent treatment with AAS mixture and 17(cid:2)-estradiol or nificantlyhigherlevelsoftestosteronederivedfromtheAASmix-
with17(cid:2)-estradiolalone. ture.Assessmentof17(cid:2)-estradiolinthemPOAindicatedthatthe
IncontrasttotheAR-deficientTfmmice,formestanetreat- amountsofthisestrogenwerecomparableincontrolwild-typeand
ment of wild-type mice neither mimicked nor enhanced AAS controlTfmanimals(p(cid:5)0.2621;Table1),consistentwithprior
effectsonGABAergictransmissionintheMPN(i.e.,prolonga- studiesexaminingperipheralestradiollevels(Vandenputetal.,2004).
tionofsynapticcurrentdecay)(Fig.2C).Specifically,therewas PreviousstudieshaveshownthatacuteexposuretotheAAS
no significant difference in (cid:7) between control wild-type mice fornon-neuronalcelllinesinvitroinhibitsaromataseactivityina
w
andwild-typemicetreatedwithformestane(21.29(cid:10)1.63msfor dose-dependent manner (Mor et al., 2001; de Gooyer et al.,
controlvs20.62(cid:10)2.04msforformestane;p(cid:5)0.8048).Similarly, 2003).TheK for17(cid:1)-MeTforinhibitionofaromatasewasfound
i
synapticcurrentdecaywasequivalentlyenhancedovercontrolin to be 0.6 (cid:3)M (Hong et al., 2008), a concentration likely to be
wild-typeanimalstreatedwithAASandinthosetreatedwithAAS attainedwiththedosesofAASadministeredhereandrelevantto
plus formestane (27.26 (cid:10) 2.00 ms for AAS-treated vs 26.62 (cid:10) humansteroidabuse(forreview,seeClarketal.,2006).Topro-
5.98 ms for AAS plus formestane; p (cid:5) 0.7933). As with AAS vide an indication of the level of aromatization (Zhang et al.,
treatment, treatment with formestane alone or concurrent treat- 2007;Zhaoetal.,2007),wedeterminedtheratioof17(cid:2)-estradiol
mentwithAASandformestanehadnoeffectonsIPSCfrequencyin totestosteroneintheintactmPOAofcontrolanimalsandani-
MPNneuronsofwild-typemice.Together,thesedataindicatethat malsreceivingchronicexposuretotheAASforbothwild-type
intermsoftheactionsofthesesteroidsonGABAergictransmission andAR-deficientTfmmice.Inbothwild-typeandAR-deficient
intheMPN,whenfunctionalARarepresent,AASactionsviatheAR genotypes,the17(cid:2)-estradioltotestosteroneratiowasmarkedly
predominateoverAASeffectsasaromataseinhibitors. lower in AAS-treated than in control subjects. Thus despite
dramaticallyelevatedlevelsofthesubstrateforaromatase,testos-
VI.Assessmentofsteroidlevelsinthebrainsof terone, parallel increases in the conversion of testosterone to
AAS-treatedmice 17(cid:2)-estradiolwerenotobservedineithergenotypeofmice.The
Previous studies have shown that adult AR-deficient Tfm mice lackofenhancedconversionoftestosteroneto17(cid:2)-estradiolin
have markedly lower levels of peripheral testosterone than AAS-treatedwild-typemicewasalsonotablegiventhatandro-
normaladultmalemice(GoldsteinandWilson,1972;Murphy genspresentintheAASmixturesignificantlyincreasedlevelsof
andO’Shaughnessy,1991,O’ShaughnessyandMurphy,1993; aromatasemRNAinthemPOA(videinfra,Fig.6).
Vandenputetal.,2004),butthatperipherallevelsofestradiol Asafinalassessmentoftestosteroneand17(cid:2)-estradiollevels
arecomparableinTfmandwild-typeTabbymales(Vandenput in the mPOA of control and AAS-treated mice, we also per-
etal.,2004).Ofparticularimportance,previousstudieshavealso formed assays for biological endpoints known to be selectively
shownthataromatizationoftestosteroneoccursintheTfmbrain dependentonAR-versusER-mediatedsignaling.Specifically,in
(Rosenfeldetal.,1977;Schleicheretal.,1986),andthatestradiol malerodents,androgensactingattheARarethemajorregulators
12492•J.Neurosci.,October7,2009•29(40):12484–12496 Penattietal.•AASEffectsintheForebrain
mRNAwas(cid:4)26timesmoreabundantthanER(cid:2)mRNAinwild-
typemiceand(cid:4)22timesmoreabundantinAR-deficientmice.
AAS treatment did not alter the levels of either ER(cid:1)or ER(cid:2)
mRNA in either wild-type (treated levels (cid:5) 105.7 (cid:10) 3.2% of
control for ER(cid:1)and 100.0 (cid:10) 5.6% control for ER(cid:2)) or Tfm
(treatedlevels(cid:5)94.2(cid:10)4.4%ofcontrolforER(cid:1)and93.7(cid:10)4.7%
controlforER(cid:2))mice(n(cid:5)8miceforeachgenotypeandeach
treatmentcondition).SubsequentWesternblotanalysisforER(cid:1)
inmPOAlysatesfromtheAR-deficientTfmmutantsrevealeda
bandof66kDa,consistentwiththeexpressionofER(cid:1)protein,
but,aswithER(cid:1)mRNAlevels,averageddensitometricsignals,
normalizedto(cid:1)-tubulin,revealednoeffectofAAStreatmenton
thelevelofER(cid:1)protein(treatedlevels(cid:5)103.5(cid:10)13.6%ofcon-
trol;n(cid:5)7control;n(cid:5)7AAS-treated).ER(cid:2)proteinexpression
Figure6. AAS-dependentchangesinAromatasemRNAinthemPOA.Dataarepresentedas
the2(cid:2)(cid:7)CTvaluesindicatingtherelativelevelsofaromatase(Cyp19)mRNAintissueisolated wasnotassessedduetolackofappropriateantibody.Together,
thesedatasuggestthatAAStreatmentdidnotelicitmarkedef-
fromthemPOAfromwild-type(left)andAR-deficientTfm(right)adultmalemiceinjectedwith
oil(control)ortreatedwiththeAASmixture(AAS)for6weeks.Identicallettersindicatemeans fectsoneitherER(cid:1)orER(cid:2)expressioninthemPOAinmiceof
thatwerenotstatisticallydifferentfromoneanotherasassessedbytwo-wayANOVAfollowed eithergenotype.
bythemeanscomparisonbyleastsignificantmeans.Analysesweremadefrom8controland8
treatedanimalsofeachgenotype. Discussion
Chronicexposureofadultwild-typemalemicetoacombination
ofaromataseexpression(Abdelgadiretal.,1994;Foidartetal., ofthreechemicallydistinctAASresultedinasignificantincrease
1995; Roselli et al., 1998) (for review, see Roselli and Resko, in AP firing with a trend toward neurons displaying irregular
1997),whileprogesteronereceptor(PR)mRNAisupregulatedin firing patterns, significant increases in the levels of (cid:1) and (cid:2)
5 1
themalemousemPOAby17(cid:2)-estradiolviaamechanismthat subunitmRNAsandaconcomitantsignificantprolongationof
requires both ER(cid:1)and ER(cid:2)signaling for maximal induction GABA receptor-mediated synaptic current decay and overall
A
(Kudwaetal.,2004).AromatasemRNAlevelswerefoundtobe charge transfer in neurons of the MPN. Acute exposure to the
significantlyhigher(p(cid:8)0.0010)inthemPOAofAAS-treated (cid:1)-selectiveantagonist,L655,708,indicatedthatreceptorscon-
5
versus control wild-type mice (Fig. 6), suggesting that the ele- tainingthissubunitareexpressedsynapticallyandextrasynapti-
vatedlevelsoftestosteronemeasuredinthemPOAmaintained callyintheMPNandthatsynapticcurrentsinL655,708-sensitive
anenhancedlevelofthisAR-regulatedtranscriptoverthetreat- neuronsareprolongedbyAAStreatment.Recombinantrecep-
mentperiod.Notsurprisingly,levelsofaromatasemRNAwere torscontaining(cid:1) subunitsarepredictedtohaveslowerdecay
5
not significantly enhanced by AAS treatment in Tfm subjects kineticsthandoreceptorscontaining(cid:1) subunits(Burgardetal.,
1
(p(cid:5)0.532)(Fig.6),consistentwiththeabsenceofclassicalAR- 1996;Dunningetal.,1999;SmithandGong,2005;cf.Pictonand
mediated signaling in this mutant mouse line. In contrast to Fisher,2007).Moreover,synapticcurrentsrecordedfromcorti-
thedramaticeffectsoftheAASmixtureonaromatasemRNA calneuronsfrom(cid:1)(cid:2)/(cid:2)micearenotablyshorter(24.8(cid:10)2.5ms)
5
inwild-typemice,noeffectsofAAStreatmentversuscontrol thanfromwild-typemice(30.3(cid:10)2.6ms)(Caraiscosetal.,2004).
on PR mRNA in the mPOA were evident in either wild-type Ourdataarethusconsistentwiththeinterpretationthatthepro-
(p(cid:5)0.2915)orTfmsubjects(p(cid:5)0.3450).Thus,thesedata longation of synaptic current observed with AAS treatment of
alsosuggestthatandrogen,butnotestrogen,levelsareelevatedin wild-typemalemiceresultedfromincreasedrepresentationof(cid:1)-
5
AAS-treatedmice.Takeninconjunctionwiththeeffectsofthe containingreceptorsatsynapsesontoMPNneurons,perhapswith
AASandagentsthatalterestrogensignalingonGABA receptor- (cid:1)-containing receptors substituting for (cid:1)-containing receptors,
A 5 1
mediatedsIPSCs(Fig.5)andtheassaysof17(cid:2)-estradioltotes- whicharelikelytobethepredominanttypeof(cid:1)subunit-containing
tosterone ratios within the mPOA (Table 1), these data are receptors expressed in the MPN of these Tabby (and Tfm) mice.
consistentwithamechanismwherebytheAASinterferewiththe Whilecomparisonswithstudiesassessingeffectsofacutelyblocking
ability of aromatase to convert androgens to estrogens in the GABAergictransmissiononAPpatterningwithinthesameneuron
intactmPOA. mustbemadewithgreatcaution,ourdatademonstratingthatlong-
termAAStreatmentresultedinenhancedGABAergictoneanda
VII.ChangesinER(cid:2)andER(cid:1)expressioninducedbychronic trend toward more irregular AP firing within the population of
exposureofAR-deficientTfmmicetoAAS MPNneuronsareconsistentwithpreviousstudiesonothercells
Whilethedatapresentedareconsistentwithanantagonisticac- types, demonstrating that acute pharmacological blockade of
tionoftheAASonendogenousestrogenproductionviainhibi- GABAergictransmissionconvertsirregulartoregularAPpatterning
tion of aromatase in the AR-deficient mice, an alternative (or (Ha¨usserandClark,1997;KononenkoandDudek,2004;Brightet
additional)mechanismtoaccountfortheseeffectswouldbeif al.,2007).
chronic AAS exposure altered the expression of either ER(cid:1)or AAStreatmentofAR-deficientTfmmicewiththisAASmix-
ER(cid:2)receptorsthemselves.Previousstudieshaveindicatedthat tureresultedindramaticallydifferentresultsfromthoseobtained
neuronswithinthemPOAofmalemiceexpressbothER(cid:1)and inwild-typemales.ChronicAAStreatmentintheAR-deficient
ER(cid:2),althoughER(cid:2)wasfoundtobeexpressedinmanyfewercells mice resulted in a significant decrease in AP frequency, no in-
than ER(cid:1)(Kudwa et al., 2004). Immunocytochemical assess- creasesineither(cid:1) or(cid:2) subunitmRNAs,butsmall,albeitsig-
5 1
mentsforER(cid:1)andthepredominantsomalGABA receptor(cid:1) nificant, decreases in (cid:2) and (cid:4) subunit mRNAs, a significant
A 3 2
isoform((cid:1))(Penattietal.,2005)indicatedthattherewasahigh decreaseinsIPSCfrequencyandamplitudeandasignificantde-
2
levelofcoincidenceofexpressionofnuclearER(cid:1)inneuronsthat creaseinGAD mRNAlevels.WhileselectiveGABA receptors
65 A
alsoexpressGABA receptors(datanotshown),andthatER(cid:1) subunitmRNAsweremodestlydecreasedintheseAR-deficient
A
Penattietal.•AASEffectsintheForebrain J.Neurosci.,October7,2009•29(40):12484–12496•12493
mice,weobservednochangesinmIPSCamplitude,kinetics,or mentwithformestane.TheAASalsopromotedasignificantde-
frequency,andourdataarethusconsistentwithamechanismby creaseinsIPSCfrequencyversuscontrolinAR-deficientmice,
which, in the absence of AR signaling, chronic AAS treatment andtherewerenodifferencesinsIPSCfrequencybetweenform-
promoted diminished AP-dependent GABA release onto MPN estaneandAAS-treatedTfmmiceorAAS-treatedTfmmiceand
neuronsandthusalsotheinhibitorydriveontothesecells.These micetreatedconcurrentlywiththeAASandformestane.Form-
dataunderscoretheideathateffectsoftheAASarewide-ranging: estanehasbeenshowntoinhibitestrogen-dependentupregula-
inbothwild-typeandAR-deficientmice,chronictreatmentwith tionofGAD andGABAproductioninhippocampalneurons
65
these steroids is likely to have significant effects not only on (Ikedaetal.,2006)andhereweshowthatGAD mRNAinthe
65
GABAergic regulation of neurons presynaptic to those studied mPOAwasdecreasedinAAS-treatedTfmmice,consistentwitha
here,butmayalsoinfluenceelectricalactivitybyalteringlevelsof
commonmechanismofaction.Finally,concurrenttreatmentof
glutamatergic drive or the function of voltage-gated channels. AAS-treatedTfmmicewith17(cid:2)-estradiolnegatedtheeffectsof
ThediversityofAASeffectsonotherthemolecularcomponents
AAStreatmentonGABAergictransmission.TheAAShavebeen
ofneuralsignalingwithinthesecriticalforebrainregionsremains
showntoinhibitaromataseactivityinvitroinnon-neuronalcell
tobeexplored.
lines (Mor et al., 2001; de Gooyer et al., 2003) and aromatase
The highly divergent actions on neuronal signaling in the
inhibition by the AAS has also been suggested to occur in fish
mPOA of wild-type versus AR-deficient Tfm male littermates
gonads(Zhangetal.,2007).Thedatapresentedhereextendthese
indicateacriticalroleforARsignalinginmediatingAASeffectsin
findingstosuggestthattheAASinhibitaromataseinthemam-
wild-type mice. The importance of AR signaling in mediating
AAS-dependentincreasesin(cid:1) mRNAexpressionandprolonga- malianbrain.TheproposedmechanismofAASinhibitionof
5 aromatasedoesnotprecludeadditionaleffectsthesesteroids
tionofsynapticcurrentdecayishighlightednotonlybyexperi-
mayhaveonotherkeysteroidbiosyntheticenzymes,suchas
mentspresentedhere,demonstratingthatsuchchangesdonot
5(cid:1)- and 5(cid:2)-reductase, that may also affect the endogenous
occurintheAR-deficientTfmline,butalsobydataindicating
levels of testosterone and estrogens. Such concurrent effects
thatcotreatmentwiththeARantagonist,flutamide,negatescom-
parable AAS-induced changes in (cid:1) mRNA levels in wild-type onotherenzymeshavebeendemonstratedforthearomatase
5
C57BL/6J mice (Penatti et al., 2009). We note that while these inhibitor,ATD(Mottaetal.,1986;Wozniaketal.,1992;Wade
datawithflutamidesuggestthatsomeoftheactionsoftheAAS etal.,1994).
reportedherearemediatedbyactivationalactionsofclassicalAR, Inwild-typeaswellasAR-deficientTfmmice,AAStreatment
they do not rule out the possibility that there may be inherent didnotresultinasignificantincreaseinthelevels17(cid:2)-estradiol
differencesbetweentheneuraltemplatesofwild-typeTabbyand orintheinductionofPRmRNA,despitedramaticallyelevatedof
Tfmmutantsthatcouldalsocontributetothedivergenceinef- testosteroneasaprecursorfor17(cid:2)-estradiol,consistentwiththe
fects of the AAS in the two genotypes. In particular, organiza- hypothesisthattheAASmayinhibitaromataseinwild-type,as
tionalactionsofandrogensactingattheARhavebeensuggested wellasAR-deficientanimals.Nonetheless,AASandformestane
withrespecttotheacquisitionofmale-specificbehaviors(Satoet didnothavecomparableeffectsontheGABAergictransmission
al.,2004;BodoandRissman,2007).SuchAR-mediatedorgani- inwild-typemice,andweconcludethatAASeffectsmediatedvia
zational effects may reflect differences in neuronal circuitry ARpredominateonthisspecificendpointinwild-typesubjects.
withinthemPOA,butthishasyetbeentested.Wealsonotethat However,thenumberofsteroid-sensitivemoleculartargetsand
mostofthewelldescribedorganizationalactionsofandrogensin steroid-sensitivephysiologicalprocessesinthebrainisextraor-
male rodents are mediated via ER following aromatization of dinarilyexpansiveandGABAergicsignalinginthemPOAisonly
circulatingandrogensto17(cid:2)-estradiol(forreview,seeSakuma, onecomponentinthisrepertoire.Thesemanymoleculartargets
2009). The diminished testosterone levels that have been re-
and their physiological outcomes are likely to be regulated by
ported for the Tfm strain are observed only in adult animals.
equivalently diverse balance between AR- versus ER-mediated
Since synthesis of testosterone (and therefore presumably also
signaling. Thus, in the wild-type male mouse, the AAS may
17(cid:2)-estradiol) is normal in perinatal Tfm mice (Goldstein and upregulate (cid:1) subunit mRNA levels and prolong GABA
Wilson,1972),manyoftheearlyorganizationaleffectsattributed 5 A
receptor-mediatedsynapticcurrentdecaybyactingasARago-
toERactivityviaaromatizedtestosteronemaythusstilloccurin
nists, but a myriad of steroid-sensitive processes yet to be
thisAR-deficientstrain.
examined may be more sensitive to an AAS-dependent de-
ExperimentsintheAR-deficientTfmlinealsosuggestanovel
crease in estrogen production. Additionally, even the same
mechanism by which the AAS may alter neuronal function in
GABAergic endpoints examined here may be preferentially
vivo.IntheabsenceofclassicalARsignaling,ourdataindicate
more sensitive to changes in ER signaling in females or ado-
that the AAS do not act, as initially expected, as ER agonists.
lescent males where both neural substrates and the endoge-
Ratherourdataindicatethatthatabuse-levelconcentrationsof
noussteroidmilieudiffersignificantly.
thesesyntheticsteroidsinterferewithERsignalingbyinhibiting
aromataseanddiminishingproductionof17(cid:2)-estradiol.Specif- Humans administer AAS in sophisticated and complex re-
gimesofdosesandpatterns,ofteninvolvingself-administra-
ically, while testosterone levels in the mPOA are dramatically
elevatedinbothwild-typeandAR-deficientAAS-treatedmice, tion of multiple classes of AAS in a pattern called “stacking”
levelsof17(cid:2)-estradiolarelowerthanincontrolsforbothgeno- (Llewellyn,2007).Ourdatahighlightthepotentialvariabilityof
types. Additionally, AAS-treated wild-type mice show signifi- molecularandcellulareffectsoftheAASinthebrainandsuggest
cantlyelevatedlevelsoftheAR-regulatedaromatasemRNA,but thattherangeofactionsofthesesteroidswillbehighlycomplex,
AAStreatmentiswithouteffectonER-regulatedPRmRNAin dependingnotonlyuponthechemicalsignaturesandconcentra-
wild-typeorAR-deficientmice.IntheAR-deficientTfmline,the tions of the different AAS taken, but also upon region-specific
AASandthearomataseinhibitor,formestane,bothsignificantly differences in AR, ER(cid:1), ER(cid:2), and aromatase expression and
decrease sIPSC amplitudes, and the AAS were without further changesintheendogenoussteroidenvironmentthatoccurasa
effectonsIPSCamplitudesinanimalsreceivingcoincidenttreat- functionofsex,age,andhormonalstate.