ContentslistsavailableatScienceDirect
Behavioural
Brain
Research
j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r
Research
report
Antidepressant-like
effect
of
the
mGluR5
antagonist
MTEP
in
an
astroglial
degeneration
model
of
depression
Helena
Domin,
Bernadeta
Szewczyk,
Monika
Wo ´zniak,
Anika
Wawrzak-Wleciał,
Maria ´Smiałowska
∗DepartmentofNeurobiology,InstituteofPharmacology,PolishAcademyofSciences,Sm˛etna12,31-343Kraków,Poland
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g
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s
•Astroglialdegenerationintheratprefrontalcortexasamodelofdepression.
•Gliotoxinl-AAAinduceddepressive-likebehaviorintheforcedswimtest.
•Antidepressant-likeeffectofmGluR5antagonistMTEPinthegliotoxinmodel.
•Imipramineantidepressantactivityinthegliotoxinmodel.
•MTEPandimipramineexertedglioprotectiveeffectsinl-AAAtreatedrats.
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Articlehistory: Received16April2014
Receivedinrevisedform8July2014 Accepted11July2014
Availableonline18July2014 Keywords: Gliotoxin l-AAA Depression mGluR5antagonist MTEP
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Theglutamatergicpredominanceintheexcitatory-inhibitorybalanceispostulatedtobeinvolvedin thepathogenesisofdepression.Suchimbalancemaybeinducedbyastrocyteablationwhichreduces glutamateuptakeandincreasesglutamatelevelinthesynapticcleft.Inthepresentstudy,wetriedto ascertainwhetherastroglialdegenerationintheprefrontalcortexcouldserveasananimalmodelof depressionandwhetherinhibitionofglutamatergictransmissionbythemGluR5antagonistMTEPcould haveantidepressantpotential.
Astrocytictoxinsl-ordl-alpha-aminoadipicacid(AAA),100g/2l,weremicroinjected,bilaterally intotheratmedialprefrontalcortex(PFC)onthefirstandseconddayofexperiment.MTEP(10mg/kg) orimipramine(30mg/kg)wereadministeredonthefifthday.FollowingadministrationofMTEPor imipraminetheforcedswimtest(FST)wasperformedforassessmentofdepressive-likebehavior.The brainsweretakenoutforanalysisondayeight.Theastrocyticmarker,glialfibrillaryacidicprotein(GFAP) wasquantifiedinPFCbyWesternblotmethodandbystereologicalcountingofimmunohistochemically stainedsections.
Bothl-AAAanddl-AAAinducedasignificantincreaseinimmobilitytimeintheFST.Thiseffectwas reversedbyimipramine,whichindicatesdepressive-likeeffectsofthesetoxins.Asignificantdecreasein GFAP(about50%)wasfoundafterl-AAA.BoththebehavioralandGFAPlevelchangeswerepreventedby MTEPinjection.
TheobtainedresultsindicatethatthedegenerationofastrocytesinthePFCbyl-AAAmaybeauseful animalmodelofdepressionandsuggestantidepressantpotentialofMTEP.
©2014ElsevierB.V.Allrightsreserved.
1. Introduction
Adecreaseinthedensityofglialcellsincorticalregions, espe-cially intheprefrontaland cingularareaswasone ofthemost consistentfindingsfrompostmortemstudiesofbrainsofdepressed patients[1–5].Moreover,adeclineinthenumberofastrocytesin
∗ Correspondingauthor.Tel.:+48126623270;fax:+48126374500. E-mailaddress:nfsmialo@cyf-kr.edu.pl(M. ´Smiałowska).
theprefrontalcortexwasobserved inratssubjected tochronic unpredictablestresswhichisoneofthegenerallyaccepted ani-malmodelsof depression [6]. Themain functionsmediatedby astrocytes includetrophic support,neuronal differentiationand synapticefficacy[7].Theyregulatethelocalconcentrationofions and neuroactivesubstances [8–12]which is crucialfor keeping thecentralnervoussystem(CNS)homeostasis.Astrocytesarean integralpartoftripartitesynapsewheretheyregulateneuronal excitability[13,14].Intheglutamatergicsynapses,themost abun-danttypeofsynapsesintheCNS,astrocytescontrolthelevelsof
http://dx.doi.org/10.1016/j.bbr.2014.07.019 0166-4328/©2014ElsevierB.V.Allrightsreserved.
extracellularglutamate,themainexcitatoryneurotransmitterin themammalianbrain,sotheymaintaintheexcitatory/inhibitory balancethere[15,16].
Ithasbeenhypothesizedlatelythatdysregulationofthisbalance underliesmooddisordersandanxietyandthattheglutamatergic predominanceinthisGlu/GABAbalanceisinvolvedinthe patho-genesisofdepression[17–20].Indeed,anincreaseinglutamateor glutaminelevelwasdocumentedinthebrainsandcerebrospinal fluidofdepressedpatients[21,22]aswellasintheirplasmaand platelets[23–25].Moreover,theinhibitionofglutamatergic func-tion had antidepressant effects [26,27]. It was also found that chronictreatmentwithantidepressantscaused thereduction of up-regulatedglutamaterelease[28–30].
Asmentionedabove, astrocyteshave a crucialrole in main-tainingtheproperGlu/GABAbalance,astheytakeupthereleased glutamate.Hence,thebasicconsequencesofthedisappearanceor dysfunctionofastrocytesincludethereductionofglutamateuptake andthus excessiveglutamatelevelsin thesynapticcleft which disturbsthebalanceandmayleadtodepression.
Recently,depressive-likebehavior hasbeendemonstrated in ratsafterdegenerationofastrocytesbygliotoxins[6].Theauthors postulatedthatastroglialdegenerationinducedbytheastrocytic toxinl-alpha-aminoadipicacid(l-AAA) in theprefrontalcortex wassufficienttotriggerdepressive-likebehavior.Previousstudies ofotherauthorsperformedin cellcultures and mice hypothal-amusshowedthat not only l-AAAbut also d-AAAand dl-AAA inducedgliotoxicdamage[31–34].Therefore,inthepresentstudy wetriedtofindoutwhethersuchgliotoxins,notonlyl-AAAbut alsodl-AAAmicroinjected intotheratmedial prefrontalcortex (mPFC), couldserve as a new animal model ofdepression and whetherourmodelofgliotoxin-induceddepressionmaybeuseful forstudyinganantidepressantactionofcompounds.Moreover,we examinedifbothtoxinsdecreasedastrocytesdensityspecifically intheprefrontalcortex.Atthebeginning,weverifiedifthe classi-calantidepressantdrug,imipramine,antagonizeddepressive-like effectsofthegliotoxinsandthenwestudiedtheeffectofa com-poundengagedintheregulationofglutamatergictransmission.
Asmentionedabove,theoveractivationofglutamatergic func-tionintheprefrontalandlimbiccorticesmaybeessentialinthe pathophysiologyofdepression[35].Itwasconfirmedbytheresults in which inhibition of glutamatergic activity by NMDA recep-torantagonistsexertedantidepressant-likeeffects inlaboratory animals[36,37]and hadantidepressantproperties indepressed patients[38–41].Ketamineisthefirstdrugofclinicalsignificance whichhasamechanismofactionassociatedwiththeNMDA recep-tor.ThisNMDAantagonistexhibitsarapidonsetofactionandhas hightherapeuticefficacy.Unfortunately,thehighantidepressant activityof ketamineis associatedwithariskof seriousadverse effects(dissociativeandpsychotomimeticeffectsandthe poten-tialtoabuse)thatgreatlyrestrictitsapplicationonalargerscale
[42].Since thetherapeutic useofdirect NMDAreceptor antag-onismisdiminishedbytheirundesirableside-effects,therefore, anindirectinhibitionofglutamatergicfunctionviametabotropic glutamatereceptors(mGluR)seemstobeamuchmore promis-ingwayfortreatmentofdepressionthanthedirectblockadeof ionotropicglutamatergicreceptors(iGluR).Indeed,mGluRligands weredemonstratedtoproduceantidepressant-likeandanxiolytic effects.MetabotropicGluRsconstituteafamilyofeightsubtypes subdividedintothreegroupsonthebasisofsequencehomology, pharmacologicalprofileand transductionpathways[43].Group I comprises mGluR1 and mGluR5 are primarilylocalized post-synaptically and are positively coupled with phosphoinositide hydrolysis, activationof phospholipase C, protein kinaseC and calciumrelease [44]. Many studies showed that stimulation of these receptors potentiated the excitatory effect of glutamate by modulation of ion channels [45], while blockade of these
receptorsattenuatedtheseexcitatoryeffects.Behavioralstudies in thelast yearshave revealed thatgroupI mGluRantagonists producedantidepressant-likeactivityinseveraltestsandmodels inrodents.Recently,MTEP (3-[(methyl-1,3-thiazol-4-yl)ethynyl]-pyridine), a highly selective, non-competitive antagonist of metabotropicglutamatereceptorssubtype5(mGluR5),hasbeen describedasanantidepressantandanxiolyticcompoundinanimal experimentsinthebehavioraldespairtestsandinthe bulbectomy-inducedmodelofdepression[46–50].Moreover,inourprevious studieswefoundneuroprotectiveeffectofMTEPwhichwasrelated to the inhibition of glutamatergic transmission and glutamate release[51].
Therefore, the goal of the present study was to find out if themGluR5antagonistMTEPmayhavepotentialantidepressant effectsintheastrocytictoxin-inducedratmodelofdepression.Such suppositionseemstobereasonableasanoveractivationof gluta-mateactivitywhichispostulatedtooccuringliotoxicmodelmay beinhibitedbyMTEP.
2. Materialsandmethods
2.1. Animals
MaleSpraque-Dawleyrats(CharlesRiver,Germany)weighing about250–300gatthebeginningoftheexperiment, wereused inthe study.Theanimalswere keptunder standard laboratory conditionsofconstant temperature(19–21◦C),controlled12:12 light-darkcycle.Theratswereage-matchedandwerehousedsix toacageona12:12light-darkcycle,withfreeaccesstofoodand tapwater.Theratsaftercannulaeimplantationwerehousedsingly. Duringtheexperiment,alleffortsweremadetominimizeanimal sufferingandtoreducethenumberofanimalsused,inaccordance withtheLocalBioethicalCommissionGuidefortheCareandUse ofLaboratoryAnimals.
2.2. Cannulaeimplantation
The rats anaesthetized with ketamine (75mg/kg i.m.) and xylazine(10mg/kgi.m.)werestereotaxically,bilaterallyimplanted withguidecannulaeaimedatthemedialprefrontalcortex(mPFC) regionusingthefollowingcoordinates:AP=+3.2mm,L=+1.0mm fromtheBregma, H=−3.5mm fromtheskull, accordingtothe Paxinos and Watson stereotaxic atlas [52]. The guide cannulae (23-gaugestainlesssteeltubing),securedbydentalcement,were anchoredtotheskullbythreestainlesssteelscrews.Inorderto preventclogging,stainlesssteelstyletswereplacedintheguide cannulaeandleftuntiltheanimalsweremicroinjected.Animals wereleftfor7daysforrecovery.
2.3. Drugtreatments
On the 1st and 2nd day of the experiment, the rats were bilaterally microinjected into medial prefrontal cortex (mPFC) withastrocytic toxins l-alpha-aminoadipic acid(l-AAA), or dl -alphaaminoadipicacid(dl-AAA)(Sigma–Aldrich ChemieGmbH, Germany).The gliotoxinswerefreshly dissolvedin0.1M phos-phatebuffer,pH7.4andweremicroinjectedatdosesof100g/2l. Control ratswere bilaterally injected with a phosphate buffer. Afterwardsdepressive-likebehaviorwasassessedbyaforcedswim test(FST)24h,72hor144hafterthesecondgliotoxinsorbuffer administrationinthetime courseexperiment,and 72h(on day 5)in all other experiments.The other groupof rats was addi-tionallytreatedwiththeantidepressantimipraminehydrochloride (Sigma–AldrichChemieGmbH,Germany)inadoseof30mg/kg, threetimes:24,5and1hbeforetheforcedswimtest(FST)orwith mGluR5antagonistMTEP(Tocris,USA)inadose10mg/kg,once
Fig.1.Experimentaldesign:scheduleoftreatmentsandtestsinthegliotoxinmodel.Arrowsindicatethetimeoftreatmentsandtestsonthetimeline.
20minbeforetheFST.Imipraminewasdissolvedinsaline,MTEP wassuspendedin0.5%methylcelluloseandbothcompoundswere administeredintraperitoneally(i.p.)ina volumeof2ml/kg.The scheduleoftreatmentisshownatFig.1.
2.4. Behavioralstudies
2.4.1. Forcedswimtest(FST)
TheratsweresubjectedtotheFSTaccordingtothemethod described inthe paperof Nowaket al. [53].Briefly, duringthe pretesttheratswereplacedindividuallyinaglasscylinders(40cm high,18cmindiameter)containing15cmofwater,maintainedat 25◦C.After15minswimmingsessionratswereremovedfromthe cylindersandreturnedtotheirhomecages.Ratswereplacedagain inthecylinder24hlaterandthetotaldurationofimmobilitywas measuredduringa5mintest.Ratswerejudgedtobeimmobile whenitremainedfloatingpassivelyinthewater.Theeffectofl -AAAordl-AAAaloneandincombinationwithimipramineorMTEP wasmeasured72hafterthesecondadministrationoftoxins.To obtainatimecourseeffectofl-AAAadministrationintheFST,the totaldurationofimmobilityinratswasmeasuredintheseparated groups24,72and144hafterthesecondl-AAAadministration.
2.4.2. Locomotoractivity
Locomotoractivitywasrecordedindividuallyforeachanimalin Opto-Varimexcages(ColumbusInstruments,USA)linkedon-line toacompatibleIBM-PC.Thebehavioroftheratswasanalyzedusing Auto-Track software (Columbus Instruments, Columbus, USA). Eachcage(43cm×43×21cm)wasequippedwitha15×15array ofinfraredemitterslocated3cmfromthefloorsurface.The num-beroflightbeamsinterruptedbyananimalwasrecordedat5min intervalsandwaspresentedasthedistancetraveledincm. Loco-motoractivitywasrecorded24or72hafterthesecondgliotoxin administration;1hafterthelastdoseofimipramineor20minafter MTEPinjection.
2.4.3. Statisticalanalysis
Theobtaineddatawerepresentedasmeans±SEM,and eval-uatedeitherbyatwo-wayANOVAanalysisofvariance,followed byBonferronimultiplecomparisontest(resultsonFigs.2and4) orStudent’st-test(resultsonFig.3).Thelevelofsignificancewas determinedasp<0.05.
2.5. Westernblotanalysis
Ondayeighth(144hafterthesecondgliotoxininjection)the ratsweredecapitated,brainsweretakenoutandtheglial fibril-laryacidicprotein(GFAP)levelwasdeterminedintheprefrontal cortex(PFC),hippocampus,amygdalaandposteriorcorticalareas. Prefrontalcortexwastakenbycuttingtheanteriorpartofthe fore-brainatthelevelofBregma2.20mmaccordingtothePaxinosand Watsonstereotaxicatlas[52].Olfactorybulbsandtheanterior stri-atumwerecutoff.Therefore,thetissuetakenforanalysiscontained majorityoftheprefrontalcortex.AdditionallyNeuNproteinlevel
Fig.2.Theeffectofcombinedadministrationofl-AAA,dl-AAAandimipramine (IMI)onthetotaldurationofimmobilityintheFSTinrats.l-AAAanddl-AAAwere administeredtwiceintotheratmPFC,atadoseof100g/2l,onthefirstandthe seconddayofexperiment.TheFSTwasperformedonday5(72hafterthesecond injection).Imipramine(30mg/kg)wasadministeredi.p.,treetimes:24,5and1h beforethetest.Theobtaineddatawerepresentedasthemeans±SEM(n=10–12 ratspergroup)andevaluatedbytwo-wayANOVA,followedbyBonferronimultiple comparisontest.*p<0.05vscontrol;#p<0.001vsl-AAAordl-AAAtreatedgroup.
(biomarker forneurons) wasestimatedafterl-AAAinjectionin thePFC.
Thetissuewashomogenizedin2%SDSandcentrifugedfor5min at10,000rpmat4◦C.Proteinconcentrationintheobtained super-natant was determinedusing a BCA Protein AssayKit (Pierce). Thesamplescontaining30gofproteinwereseparatedby10% SDS-polyacrylamidegelelectrophoresisandtransferredto nitro-cellulose membranes using an electrophoretic transfer system. Non-specificbindingwasblockedfor30mininblockingreagent (Roche).Then,membraneswereincubatedovernightat4◦Cwith mousemonoclonalanti-GFAPantibody(1:5000,Millipore)or anti-NeuN(1:1000,Millipore).Themembraneswerethenwashedwith TBS-Tand incubatedfor 30minwitha horseradish peroxidase-conjugatedanti-mouseIgGsecondary antibody(1:1000,Roche). ThereactionwasvisualizedbyusingBMChemiluminescence West-ern BlottingKit, Roche). Chemiluminescence wasrecorded and evaluatedwithaluminescentimageanalyzer(FUJI-LAS-4000).The
Fig.3. Theeffectofl-AAAadministrationonthetotaldurationofimmobilityinthe FSTinratsatdifferenttimepoints.l-AAAwasadministeredtwiceintotheratmPFC atadoseof100g/2l,onthefirstandtheseconddayofexperiment.TheFSTwas performed24,72and144hafterthesecondtoxininjection.Theobtaineddatawere presentedasthemeans±SEM(n=10–12ratspergroup)andevaluatedbyStudent’s t-test.Significancewasassumedatp<0.05.
Fig.4.Theeffectofcombinedadministrationofl-AAAandMTEPonthetotal dura-tionofimmobilityintheFSTinrats.l-AAAwasadministeredtwiceintotheratmPFC atadoseof100g/2l,onthefirstandtheseconddayofexperimentandMTEP, 10mg/kg,i.p.,wasgiven20minbeforetheFST.Theobtaineddatawerepresented asthemeans±SEM(n=10–12ratspergroup)andevaluatedbytwo-wayANOVA, followedbyBonferronimultiplecomparisontest.*p<0.05vscontrol;#p<0.001vs
l-AAAtreatedgroup.
relativelevelsofimmunoreactivitywerequantifiedusingImage Gaugev4.0software.Toconfirmequalloadingofthesampleson thegel,theblotswereincubatedwithmouseanti--actin anti-body(1:5000,Sigma)andthenprocessedasdescribedabove.The densityofGFAPproteinbandwasnormalizedtothedensityofthe -actin.
TheWestern blotanalysis ofGFAP was performedin group of rats treated with l-AAA, dl-AAA, l-AAA+imipramine, dl -AAA+imipramine,imipramine,MTEPand l-AAA+MTEP.Forthe timecourseeffectofl-AAAonGFAPandNeuNproteinlevelinthe PFC,ratsweredecapitatedandthebrainswerecollected24,48and 72hafterthesecondinjectionofgliotoxin.Westernblotanalysis wasperformedasdescribedabove.
2.5.1. Statisticalanalysis
Theobtaineddatawerepresentedasmeans±SEM,and eval-uatedeitherbyatwo-wayANOVAanalysisofvariance,followed byBonferronimultiplecomparisontest(resultsonFigs.6and7) orStudent’st-test(resultsonFig.5).Thelevelofsignificancewas determinedasp<0.05.
2.6. Histologicalandimmunohistochemicalstudies
Ondayeighth(144hafterthesecondinjectionofthetoxin) thebrainsweretakenforanalysis.Ratsunderdeeppentobarbital anesthesiawereperfusedthroughtheascendingaortawith0.9% physiologicalsaline,followedbyacold4%paraformaldehyde(PF) in0.1Msodiumphosphatebuffer,pH7.4.Next,theirbrainswere removed,postfixedincoldbufferedPFfor3h,andcryoprotectedin abuffered20%sucrosesolutionforatleast5daysat4◦C.Thebrains werethenfrozenondryice,and40mcoronalsectionswerecutat levelscontainingmedialprefrontalcortex(betweenbregma4.70 to1.70mm,accordingtothePaxinosandWatsonstereotaxicatlas
[52].Free-floatingsectionswerecollectedandeverysixthsection weretakenforglial fibrillaryacidic protein(GFAP) immunohis-tochemicalstaining,usinganti-GFAPmousemonoclonalantibody (Millipore)dilutedat1:800insodiumphosphate-buffer(PBS) con-taining0.2%TritonX-100and1%normalhorseserum.Adjacent sectionswerestainedwithcresylvioletandusedforahistological analysisandverificationoftheinjectionsites.Theimmunostained sections,afterreactionwithprimaryantibodywererinsedinPBS andprocessed byan avidin–biotinperoxidasecomplexmethod usinganABC-peroxidasekit(Vector Lab)anddiaminobenzidine
Fig.5.Theeffectofl-AAAadministrationontheGFAPlevelintheratPFC,measured atdifferenttimepoints.Westernblotanalysis.l-AAAwasadministeredtwiceinto theratmPFCatadoseof100g/2l,onthefirstandtheseconddayofexperiment. TheGFAPlevelintheprefrontalcortex(PFC)wasdetermined7h,24h,48h,72h and144hafterthesecondtoxininjection.(A)Theimmunoblotbandscorresponding toGFAP(upperpanels)and-actin(lowerpanels)areseen.Aweakerintensityof theGFAPbandisobservedinsamplestakenfromrats72and144hafterthel-AAA injection.(B)AnalysisofdataoftheGFAPlevel.Theobtaineddatawerepresented asthemeans±SEM(n=6–7ratspergroup)andevaluatedbyaStudent’st-test. *p<0.05,**p<0.01vscontrol.
(DAB)as a chromogen.The stained sectionswere mounted on slides,dried,dehydrated,clearedinxylene,cover-slippedwith Per-mountandanalyzedunderalightmicroscope.
Additionally,somesectionsfromeachgroupwerestainedby immunofluorescencemethodusinganti-GFAPantibody.After1h incubation in a blocking buffer(5% normal donkey serum and 0.2% Triton X-100 in 0.01MPBS) the sections were incubated (48hat4◦C)withtheprimaryanti-GFAPmouseantibody (Mil-lipore)diluted at1:800in theblockingbuffer. Afterincubation withtheprimaryantibody,thesectionswerewashedand incu-batedovernightat4◦CinthesecondaryantibodyCy3-conjugated anti-mouseIgG(1:200,JacksonImmunoResearch,USA).The sec-tionswerethenwashedandmountedwithacoverslipoverlay.The
Fig.6.Theeffectofl-AAAandimipramine(IMI)administrationontheGFAPlevel intheratPFC.Westernblotanalysis.l-AAAwasadministeredtwiceintotherat mPFCatadoseof100g/2l,onthefirstandtheseconddayofexperiment.The GFAPlevelwasdeterminedonday8(144hafterthesecondtoxininjection).(A) Theimmunoblotbands.(B)AnalysisofdataoftheGFAPlevel.Theobtaineddata werepresentedasthemeans±SEM(n=6–7ratspergroup)andevaluatedby two-wayANOVA,followedbyBonferronimultiplecomparisontest.*p<0.05vscontrol;
Fig.7. Theeffectofl-AAAandMTEPadministrationontheGFAPlevelintherat PFC.Westernblotanalysis.l-AAAwasadministeredtwiceintotheratmPFCata doseof100g/2l,onthefirstandtheseconddayofexperiment.TheGFAPlevel wasdetermined144hafterthesecondtoxininjection.(A)Theimmunoblotbands. (B)AnalysisofdataoftheGFAPlevel.Theobtaineddatawerepresentedasthe means±SEM(n=6–7ratspergroup)andevaluatedbytwo-wayANOVA,followed byBonferronimultiplecomparisontest.*p<0.05vscontrol;#p<0.05vsl-AAA.
sectionswereanalyzedwithaconfocallaserscanningmicroscope, DMRXA2TCSSP2(Leica),witha63×/1.40–0.70oilobjective(Leica) drivenbyconfocalsoftware(Leica)usingthesequentialscan sett-ings,asdescribedpreviously[54].WorkingwithGreNelaserline emittingat543nmwasusedtoexciteCy3-conjugatedantibody. Thebackgroundnoiseofeachconfocalimagewasthenreducedby averagingfourscans/lineandsixframes/image.Thepinholevalue ofoneairywasusedtoobtainflatimages.
2.7. StereologicalcountingofGFAP-immunoreactiveastrocytes
The number of GFAP-immunoreactive astrocyte cell bodies stainedwithDAB,inthemedialprefrontalcortex(mPFC)was eval-uatedbystereologicalcounting.Theprocedureswereperformed usingamicroscope(Leica,DMLB;Leica,Denmark)equippedwith aprojectingcamera(BaslerVisionTechnologies,Germany)anda microscopestage connectedtoan xyzstepper (PRIORProScan) controlledbya computerusingVisiopharm NewCASTsoftware (Visiopharm,Denmark).
Systemic uniform, random sampling was used to choose thesectionsfor theanalysis.For stereologicalestimates, GFAP-ir astrocytes were counted within the contours of the mPFC [AP=4.70–1.70mm]from bregmaaccordingtothePaxinos and Watsonstereotaxicatlas[52]inatleast8–12sectionsat240m intervals.ThemPFCwasoutlinedunderalowermagnification(5×) anduniformlysampleddissectorpointswereusedatrandomalong thewholestructureusingthemeandersampling.
The total number of GFAP-positive cells in the mPFC was unbiasedly estimated using a three dimensional probe under a higher magnification (63×) according to the formula: N=Q×V(ref)/
v
(dis)×P,whereQisatotalcountofGFAP-ir astrocytesintheuniformlysampleddissectors,V(ref)–thetotal volumeof themPFC,v
(dis)– thetotal volume ofthe dissector[55]andP–thetotal numberofalldissectorpoints.A count-ingframeof74.61m×74.61m(5567.27m2),asamplinggrid
of333.68m×333.68m(111,342.3m2)andadissectorheight
of20mbelowthesurfacewereemployed.Samplingwas opti-mizedtoproduceacoefficientoferrorwellundertheobserved biologicalvariability[56].ThetotalvolumeofthemPFCV(ref)was estimatedusingCavalieri’sprinciple[56]accordingtotheformula:
V(ref)=t×a(p)×P,wheretistheknowndistancebetweenthe sampledsections,a(p)istheareaassociatedwitheachpointofa grid,Pisthetotalnumberofcountedpointsoverallsectionsfrom onerat.
2.7.1. Statisticalanalysis
Thedatawerepresentedasmeans±SEM,andevaluatedbya one-wayANOVAanalysisofvariance,followedbyTukey’s multi-plecomparisontest.Thelevelofsignificancewasdeterminedas p<0.05.
3. Results
3.1. Behavioralstudies
3.1.1. Effectofcombinedadministrationofl-AAAordl-AAAand imipramineintheFST
Theeffectof l-AAAand dl-AAAonthetotal durationof the immobility time in the FST in rats is shown in Fig. 2. l-AAA and dl-AAA administered twice into the rat mPFC, at a dose of100g/2l,inducesdepressive-like behaviorin theFST.The immobilitytimeofratsincreasedsignificantlybothinl-AAAand dl-AAAtreatedgroupascomparedtocontrolgroup. Administra-tion of imipraminesignificantly decreased the immobilitytime of rats and antagonized the effects elicited by l-AAA and dl -AAAadministration. Twoway ANOVAdemonstrated significant effectofgliotoxin[F(2,47)=3.32,p=0.0448];significanteffectof imipramine[F(1,47)=83.87,p=0.0001]andsignificantinteraction [F(2,47)=4.03,p=0.0242].
3.1.2. Effectofl-AAAintheFSTatdifferenttimepoints
Thetime courseeffectofl-AAAonthetotal durationof the immobilitytimeintheFSTinratsisshowninFig.3. Depressive-likebehavior,observedasincreasedimmobilitytime intheFST wasfound 24h(p=0.0445) and 72h(p=0.0491) but not 144h (p=0.5103)afterthesecondgliotoxininjection.
3.1.3. Effectofcombinedadministrationofl-AAAandMTEPin theFST
The effect of combined administration of l-AAA and MTEP on the total duration of the immobilitytime in FST in rats is showninFig.4.l-AAAadministeredalonesignificantlyincreased the immobility time in FST. MTEP given alone significantly decreasedtheimmobilitytimeinratsandantagonizedtheeffect induced by l-AAA.Two way ANOVA demonstrated the signifi-canteffectofMTEP[F(1,39)=13,49,p=0.0007],significanteffect of l-AAA [F(1,39)=5.44, p=0.0249] and significant interaction [F(1,39)=4.21,p=0.047].
3.1.4. Effectofl-AAAanddl-AAAaloneorincombined administrationwithimipramineandMTEPonthelocomotor activityinrats
Theeffectofl-AAAanddl-AAAadministrationaloneorin com-bined treatment withimipramine and MTEPon thelocomotor activityinratsisshowninTable1.
l-AAA anddl-AAAadministered alone didnotinfluence the locomotor activityinrats (Table1A).However,theimipramine alone or in combined administration with l-AAA and dl-AAA significantlydecreased thelocomotor activityin rats,measured 72hafterthesecondinjectionoftoxin(Table1B).Nosignificant effectonthelocomotoractivitywasfoundafterMTEPand com-binedl-AAAand MTEPinjections.Onlyatendencyfordecrease was observed (Table 1C). Two way ANOVA demonstrated no effectof gliotoxinsF(2,25)=1.01, p=0.3802,significanteffectof
Fig.8.MicrophotographsofcoronalsectionsoftheratbrainmPFC.(A,B,CandD)Cresylvioletstaining.Generalviewofthesectionsofcontrol(A)andl-AAA(B)treatedrats underlowermagnification.Glialscar(inA)andtissuedestruction(inB)isseenintheinjectionsites.Blacksquarespointtheregionspresentedunderhighermagnification onCandDrespectively.Stainednervecellbodiesareseenneartheinjectionsitebothinacontrolrat(C)andinanl-AAAinjectedrat(D).Glialscarisvisibleatthebottom ofthephotographs.(EandF)SectionsimmunostainedwithGFAPantibody.NumerousGFAPpositiveastrocytesareseeninthesectionfromacontrolrat(E),incontrastto nearlynoastrocytesinthesectionfromanl-AAAtreatedrat(F).CalibrationbarsforAandB500m,for(C),(D),(E),(F)25m.(Forinterpretationofthereferencestocolor inthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)
imipramineF(1,25)=56.50,p<0.0001andnotsignificant interac-tionF(2,25)=0.15,p=0.8642.
3.2. Westernblotanalysis
3.2.1. Effectofl-AAAadministrationontheGFAPandNeuN proteinlevelintheratPFCatdifferenttimepointsandindifferent structures
Thetimecourseeffectofl-AAAontheGFAPproteinlevelinrat PFCisshowninFig.5.SignificantdecreaseintheGFAPproteinlevel wasobserved72h(p=0.0298)and144h(p=0.0195)aftersecond l-AAAadministration.NochangesintheGFAPproteinlevelwere observed7,24and48hafterl-AAAtreatment.l-AAAinjectiondid notinfluencetheNeuNlevelinPFCatanytimepoints(datanot shown).NochangesintheGFAPlevelwerefoundintheposterior
corticalareas,hippocampusandamygdala(datanotshown)after thetoxinadministration.
3.2.2. Effectofl-AAA,dl-AAAandimipramineadministrationon theGFAPproteinlevelintheratPFC
Theeffectsofl-AAAaloneandcombinedadministrationofl -AAAwithimipramineontheGFAPproteinlevelinPFC(144hafter thetoxininjection)areshowninFig.6.
l-AAA administration induced a significant reduction in the GFAP protein level in the rat PFC (to 46% of control level). Imipraminealone hadnoeffectonGFAPprotein levelhowever antagonizedtheeffectinducedbyl-AAA.TwowayANOVA demon-strated no effect of l-AAA [F(1,21)=2.18, p=0.1544]; no effect of imipramine [F(1,21)=0.33, p=0.569] and significant interac-tion[F(1.21)=8.85,p=0.007].Nosignificantchangeswerefound
Table1
Theeffectofl-AAAanddl-AAAaloneorincombinedadministrationofl-AAAand MTEPonthelocomotoractivityinrats.
Treatment Activitycounts(5mintest)
A Control 662.08±66.1 l-AAA 670.37±32.5 dl-AAA 693.79±49.3 B Control 583.7±10.08 Imipramine 213.9±43.03a l-AAA+imipramine 311.5±81.43a dl-AAA+imipramine 212.9±28.25a C Control 622.5±64.99 MTEP(10mg/kg) 466.54±37.19 l-AAA+MTEP(10mg/kg) 487.5±45.55
Locomotoractivitywasexamined;(A)24hafterthesecondl-AAAordl-AAA admin-istration;(B)72hafterthesecondadministrationofl-AAAand1hafterthelastdose ofimipramine(triplicateinjectionsofimipramine24,5,1hbeforethetest);(C)72h afterthesecondadministrationofl-AAAand20minaftertheMTEPtreatment.The valuesrepresentmean±SEM(n=6–8ratspergroup).
ap<0.05vscontrol.
afterdl-AAAaloneandincombinationwithimipramine(datanot shown).
3.2.3. Effectofcombinedadministrationofl-AAAandMTEPon theGFAPproteinlevelintheratPFC
Theeffectsofl-AAAaloneandtheeffectofcombined adminis-trationofl-AAAwithMTEPontheGFAPproteinlevelinPFC(144h afterthetoxininjection)areshowninFig.7.
l-AAAadministrationinducedsignificantreductionintheGFAP proteinlevelintheratPFC(to52,8%ofcontrol).MTEPantagonized theeffectinducedbyl-AAAbutdidnotchangetheGFAPprotein levelwhengivenalone.TwowayANOVAdemonstratednoeffectof l-AAA[F(1,30)=0.75,p=0.3523];noeffectofMTEP[F(1,30)=0.44, p=0.5102]andsignificantinteraction[F(1.30)=7.54,p=0.0101].
3.3. Histologicalandimmunohistochemicalanalysis
3.3.1. Effectofl-AAAanddl-AAAaloneorincombined administrationofl-AAAandMTEPonthenumberof GFAP-positivecells
Histological analysis of sections stained with cresyl violet showedtheproperlocalizationoftheinjectionsites(inmPFC), and CVstainedneuronswereseen inthewholemPFC (Fig.8A andB), alsoveryneara scar(Fig.8Cand D)GFAP immunohis-tochemistryshowedadecreaseinadensityofastrocytesinthe mPFC of rats treated with l-AAA (Fig. 8E and F). Stereological countingshowedastrong,significantdecreaseinthenumberof GFAP-immunoreactive(ir)astrocytesinmPFCafterl-AAAin com-parisontocontrolrats(to54,18%ofcontrol)(Fig.9A).Theeffectof dl-AAAonthenumberofGFAP-irastrocyteswasweakand insignif-icant.Thedecreasingeffectofl-AAAonthenumber ofGFAP-ir cellswassignificantlyinhibitedbyMTEP,reaching81,2%ofcontrol (Fig.9B).TheglioprotectiveeffectofMTEPfoundinstereological countingwasconfirmedbythemorphologicalobservationof astro-cytesinthemPFCimmunostainedwithananti-GFAPantibody.A densityofGFAPimmunofluorescentastrocytesdecreasedbyprior l-AAAadministrationwasnearlynormalizedafterMTEPtreatment (Fig.10).
4. Discussion
Our results indicate that gliotoxin induced degeneration of astrocytesin therat medialprefrontalcortexmaybeusedasa
Fig.9.Theeffectofl-AAAanddl-AAAadministration(A)andtheeffectofcombined administrationofL-AAAandMTEP(B)onthenumberofGFAPpositivecellsinthe ratmPFCcountedbystereologicalmethod.l-AAAwasadministeredtwiceintothe ratmPFCatadoseof100g/2l,onthefirstandtheseconddayofexperimentand MTEP10mg/kg,i.p.,wasinjectedonday5(20minbeforeFST).Brainsweretaken foranalysisonday8(144hafterthesecondtoxininjection).Thedataarepresented asthemeans±SEM,(n=5–8ratspergroup)andevaluatedbyone-wayANOVA, followedbyTukey’smultiplecomparisontest.p<0.05wasconsideredsignificant. **p<0.01vscontrol;#p<0.05vsl-AAA.
good experimentalmodel ofdepression. The resultsarein line withtheideaarisingfromnumerousstudiesindicatingthat astro-cytedysfunctionplaysacrucialroleinpathogenesisofdepression. Actually, anhedonia wasobserved in ratsafter theblockade of astrocyticglutamateuptakeintheprefrontalcortex[57]. Intracere-broventricularinfusionofdihydrokainate,anastrocyticglutamate uptake blocker,alsoinducedsomesignsofanhedoniaobserved intheintracranial stimulationandplaceconditioningtests[58]. Theblockadeof astrocyticglutamateuptakein theamygdala, a limbicstructureengagedintheregulationofmoodandanxiety hasbeenreportedtoinducesomebehavioraleffectscharacteristic ofdepressive-likesymptoms[59].Reducedglutamatetransporter activityandexpressionwasreportedtooccurinthehippocampus inthemousestressmodelofdepressionandinthecortexinthe learnedhelplessmodelinrats[60].Alsoinhumanpostmortem studies,downregulationofgenescodingforastrocyticglutamate transporterswasobservedinthecingulateandprefrontalcortexof depressedsubjects[61].Dysfunctionofastrocytegapjunction com-municationwasalsofoundintheratprefrontalcortexafterchronic unpredictablestressanditwasreversedbytreatmentwith antide-pressants,moreover,thepharmacologicalgapjunctionblockade inthePFCinduceddepressive-likebehaviorinrats[62].Allthese findingsindicatethatdamageorimpairmentofproperfunctionof astrocytesintheprefrontalcortexmaybeagoodmodelof depres-sion.
Inourstudies,wedamagedastrocytesintheratmPFCbythe gliotoxin,alpha-aminoadipicacid(AAA).Itwasdemonstratedtobe aspecifictoxinforastrocytesalreadyin1971byOlneyand cowork-ers[63],anditsspecificitywasconfirmedinlateryearsbothinvivo
Fig.10.MicrophotographsofcoronalsectionoftheratbrainmPFC,showingGFAPpositiveastrocytesstainedbyimmunofluorescencemethod.(A)Numerousastrocytesare seeninthesectionfromacontrolrat;(B)thedecreaseinastrocytedensityanddiminutionoftheirprocessesarefoundafterl-AAAinjection;(C)MTEPpartiallypreventsthe l-AAAeffect:numerousastrocytesarevisiblelikeinthecontrolsection.
andinvitro[32,33,64].Itistakenupspecificallybyastrocytesand accumulatinginthesecellsexertsitsgliotoxiceffectbyinterference withproteinsynthesis[31,34,65].
Studies of other authors concerning the effects of d- and l-isomers or racemic form of AAA gave conflicting, heteroge-neousresults.Whenadministeredsubcutaneouslyintoinfantmice, it exerted a toxic effects in thearcuate hypothalamic nucleus, whereas d-AAA and dl-AAA induced, respectively, a mild to extremegliotoxicbutnotneurotoxicdamageandl-AAAinduced notonly gliotoxicbut alsosomeneurotoxic changes,moreover d-isomerhadevenanti-neurotoxicproperties[32,64,66].Similar resultswereobtainedinastrocytesormixedneuronal/astrocyte cultures [33] but additionally d-isomer appeared to be toxic onlyfor mitoticcells [31]. Theeffectof AAAonastrocyteswas alsostudiedinadultratsafterintracerebralinjectioninto differ-entbrainstructures.Asinglel-AAAinjectionintotheamygdala induced loss of astrocytes and their markers (GFAP, S-100) in a wide areaaroundthe injectionsite, while neuronsremained undamaged[67].Astroglialdegenerationwasalsoobservedafter l-AAAinjectionintothestriatum[68],substantianigraandlocus coeruleus[69].OntheotherhandSaffranandCrutcher[70]failedto observegliotoxicpropertiesofl-AAAordl-AAAinthe hippocam-pus and striatum afterinjections into those structures. Allthe abovementionedstudies,werefocusedonlyonthecellulareffects ofthetoxins,andtheauthorsdidnot examinetheirbehavioral effects.
Inourstudiesweusedl-AAAanddl-AAAasamodelof depres-sionbasedonastrocyteablationinthemedialprefrontalcortex (mPFC).SuchmodelwasdevelopedbyBanasrandDuman[6].The authorsdemonstrated thata double microinjectionof gliotoxin l-alpha-aminoadipicacid(l-AAA)intotherat medialprefrontal cortexinducedastroglialdegenerationintheprefrontalcortexand triggeredadepressive-likebehaviorsimilartothatobservedafter chronicunpredictablestress,agenerallyacceptedmodelof depres-sion.Dosesoftoxinandgeneralscheduleofourexperimentwere similarbutweusednotonlyl-AAAbutalsodl-AAA,asthisracemic formhasbeensuggestedbysomeauthorstobemorespecificfor astrocytes(asalreadymentionedabove).
Ourpresentresultsshowedthatbothgliotoxinsl-AAAanddl -AAAmicroinjected into the medial prefrontalcortex of the rat induceddepressive-likebehavioraleffects,becauseanincreasein immobilitytimeintheforcedswimtest(FST)withoutchanging locomotoractivitywasobservedafterthesetoxins.Theeffectwas observed24and72hafterthesecondgliotoxininjectionbutnot after144h.TheresultsareinagreementwiththefindingsofBanasr andDuman[6]whodemonstratedl-AAA-induceddepressive-like behavioralchangesinratsinthesucrosepreferencetestandforced swim test,in thenovelty suppressedfeedingmodel and active avoidancetestatsimilartimepoints,buttheydidnotstudythe
behavioraleffectafter144h. Wefoundthatnotonlyl-AAAbut alsodl-AAA revealed similar depressive-like action and, more-over,theeffectwaspreventedbytheclassicalantidepressantdrug, imipramine,usedindosesandscheduletypicalforsuchstudies
[71].Theseresultsindicatethattheintra-mPFCgliotoxininjection maybeagoodmodelforstudyingantidepressanteffectsofdrugs.
Weverifiedalsotheeffectsofthegliotoxinsonastrocytesby studyingtheappearanceandthelevelofGFAP,aprotein character-isticofastrocytes[72],usingimmunohistochemicalandWestern blotanalysis.BothmethodsshowedadecreaseinGFAP-irinthe PFCafterl-AAAmicroinjection,whichindicatedadamageof astro-cytes.TheeffectisinlinewiththeresultsofBanasrandDuman[6]
whofoundadecreaseindensityofGFAPpositivecells144hafter l-AAAinchosenfieldsoftheprefrontalcortex.dl-AAAdisplayeda tendencytowarddecreasingoftheaboveparametersbuttheeffect wasstatisticallynon-significantprobablybecauseofhigh variabil-ityoftheresults.Forthatreason,wehavechosenl-AAAforfurther studies.
TheWesternblotanalysisofthetime-courseofl-AAA-induced changesshowedaprogressivedecreaseinGFAPleveldisplaying anon-significantlowering48handastrongsignificantdecrease 72and144hafterthesecondtoxininjection.Nosuchtime-course studieswereperformedearlier.Itisdifficulttoexplainthe discrep-ancybetweenlowGFAPlevel144hafterthetoxinandthelackof depressive-likebehavioraleffectintheFSTatthesametimepoint. Wemayhypothesizethatsomecompensatorychangesmighttake placeafterthelongertimeperiod,eveninotherbrainstructures, whichinfluenced animal behavior.Thishypothesis seemstobe supportedbyourpreliminaryfindingswhich showedthatGAD (glutamicaciddecarboxylase)proteinlevel,amarkerofGABA neu-rons,increasedinthehippocampus144hafterthel-AAAinjection (ourunpublishedobservations).Asdepressionispostulatedtobea resultofGABA/Gluimbalancewithglutamatergicoveractivity(see introduction),theactivationofGABAfunctionmayhavepositive antidepressanteffectsonbehavior.
We verified whether our gliotoxin model may be a useful modelofdepression.Forthispurposeweadministeredimipramine, a standard antidepressantdrug,tol-and dl-AAAinjected rats. Imipraminewasgivenintripledoseswhichisroutinetreatment forscreeningantidepressiveeffectsinFST(accordingto[73,74,75]). Weevidencedthatimipraminereversedthebehavioraleffectsof bothl-AAAanddl-AAA.Thetimeofimmobilitydecreasedeven belowthecontrollevel.Theimipramineeffectseemstobe specif-icallyconnectedwithanantidepressantaction,becausethedrug decreasednotincreased,thelocomotoractivity.
Wealsostudiedwhetherimipraminepreventedthegliotoxin induced astrocyticdegeneration in thePFC using Western blot method.While imipramine reversed decreasingeffectof l-AAA on GFAP level, the effect of dl-AAA was only weakly and
non-significantlyinfluenced(datanotshown).Summingup,l-AAA toxinseemstobemorepromisingasamodelofdepression.
Therearenostudiesyetontheeffectofimipramineorother antidepressantsinthegliotoxicmodelboth onbehaviorand on astrocyte density and GFAP protein level. However, in another model,chronicunpredictablestressinrats,bothastrocyte pathol-ogyanddepression-likebehaviorwerereversedbyantidepressants clomipramine, fluoxetine and magnolol [76–79]. The effect of antidepressanttherapyonastrocyteswasdemonstrated bothin experimentalstudiesandindepressedpatients[80].
Wealsoexaminedwhetherthetoxinsusedinourexperiments, l-AAAanddl-AAA,killedneuronsbesidesastrocytes.Wedidnot observeneuronal loss in histologicaland Westernblotstudies. Nodiminutionofnerve cellsdensitywasseeninbrainsections fromthemPFC,stainedwithcresylviolet.Nervecellbodieswere observedevennearthecannulaetrackandglialscar(Fig.8Aand B).WesternBlotanalysisofNeuNproteinlevel wasfocused on theeffectofl-AAAonly,because,asmentionedabove,Lisomer ofAAAwaspostulatedbysomeauthorstohavesomeneurotoxic effects[32,33].NochangesinNeuNlevelwerefoundduring7h to144hafterthetoxininjection.Thisresultisinagreementwith theobservationsofBanasrandDuman[6]whofoundno notice-ableneuronallossintheprefrontalcortexafterl-AAAinsections stainedimmunohistochemicallywithNeuNantibody.
As mentioned in the introduction, the glutamatergic over-activityhas been postulatedto be essential in pathogenesis of depression,andthemodelofastrocyteablationisbasedonthe dys-regulationoftheGlu/GABAbalance.Therefore,inthepresentstudy, weusedpotentialantidepressantcompound, MTEP,toexamine whetheritcanpreventthedepressive-likeeffectsofl-AAAtoxin,as measuredbybehavioraltestandastrocytedegenerationmarkers. MTEPisahighlyselectivenegativeallostericmodulatorofmGlu5 receptors[81–83].
Antidepressant-likeeffectofMTEPwasobservedin previous studies in rat and mouse models. Pałucha et al. [47] showed thatasingleintraperitonealinjectionofMTEPproduceda signif-icant,dosedependentdecrease in theimmobilitytime ofmice inthetail-suspentiontest(TST)andchronictreatmentwiththis compound inhibited bulbectomy induced hyperactivity in rats. Antidepressant-likeeffectsofMTEPandothermGluR1andmGluR5 antagonistswerealsodemonstratedintheforcedswimtestinrats andtheTSTinmice[84–86].Similarly,theothernegativeallosteric modulatorofmGluR5,GRN-529,givenorally,1hbeforethetest,in asingledoseintomiceinducedantidepressant-likeandanxiolytic effects[83].
Ourresultsindicatethat MTEPpossessesantidepressant-like activityalsoin thegliotoxininduced modelof depression.This compoundinjectedonce20minbeforetheforcedswimtest(FST), preventedthel-AAAinducedincreaseinimmobilitytimeofrats. Italsoreversed thedecreasingeffectofl-AAAonGFAP levelin PFC,measured144hafterthetoxinusingWesternblotmethod, andonthenumberofGFAP-ircellsdeterminedbystereological counting.
Itisespeciallyinterestingthattheantidepressant-likeeffectof MTEPwasfoundafterasingleinjection.Tillnow,mostofthestudies concerningantidepressantdrugsdescribedthattheywereeffective afterchronictreatmentonly,bothinhumanandanimal experi-mentalstudies[87,88],butthosestudieswerefocusedmainlyon thedrugsactingthroughthemonoaminergicmechanismsandthey neededweeksormonthsfordevelopingtheirtherapeuticeffects. Moreover,about30–50%ofpatientsremainsresistanttotreatment
[89].Ontheotherhand,thecompoundsactingbyinhibitionof glu-tamatergicactivity,likeNMDAreceptorblockers(i.e.dizocilpin), revealedtheirantidepressantactivityquicklyafterasingledose
[90,91].MetabotropicGlu5receptorsareanatomicallyand func-tionallycoupledwithNMDAreceptors[92–94]andtheiractivation
potentiatesNMDAreceptor activity,sothemGluR5 antagonists indirectlyinhibitthem[95].
MTEPcounteractednotonlythedepressive-likebehaviorafter l-AAAbutalsopreventedtheastrocyticdegenerationsinthemPFC inducedbythetoxin.Themechanismofsuchprotectiveeffects isnotclearbutitmaybeconnectedwithmGluR5inhibitionand in consequence witha diminution of NMDAreceptor function. NMDAreceptors arepresent onastrocytes and otherglial cells
[96].MetabotropicGlu5receptorsareveryabundantinastrocytes
[97–101] and are found tobe connected with NMDA receptor currents [102–104]. Inhibitionof mGluR5 and NMDA receptors hasneuroprotectiveand glioprotectiveeffects [51,105,106].Itis worthwhile noting that l-AAA-induced astrocytic degeneration developedgradually(seeresults,Fig.5),andat72hafterthetoxin, whenMTEPwasinjected,thelevelofGFAPwasstillmaintained atabout50%,sothedrugcouldaffecttheseremainingastrocytes. Manystudieshaveshownthatantidepressantsstimulate prolifer-ationofastrocytesandproductionoftrophicfactorsbythesecells, suchasBDNF,GDNF,FGFandothersmodulatorswhichmay resti-tute properneuroglia networkand theirfunction[79,107–111]. Moreover,sucheffectsmayappeareven afterashorttreatment and a single dose. The first molecularresponses in ERK/MAPK signalingpathwayin astrocytesmayappear alreadyafteracute administration ofdesipramineor fluoxetine[110].Primary cul-turesofratastrocytestreatedwithfluoxetinerevealedinductionof MAPK,BDNF,GDNFandtheirreceptorgenesalready2hafterthe beginningofthetreatment[112].Especiallyfasteffectswerefound afterthecompoundsactingthroughtheglutamatergicreceptors, butmostof thosestudiesinvestigatedonly behavioralchanges, orreceptorresponses[113,114].Inclinicalstudies,rapid(during 2h)antidepressanteffectswereproducedbytheNMDAreceptor antagonistketamineorthelow-trappingNMDAchannelblocker AZD6765[26,115].Thebehavioralantidepressant-likeeffectswere alsorevealedsoonafterasinglemGluR5antagonisttreatment(as mentionedabove).
Therefore we can hypothesize that antidepressant-like and antigliotoxiceffectsofMTEPdemonstratedinthepresentgliotoxic model of depression, may result from its inhibitory action on mGluR5andindirectlyonNMDAreceptorsboth inneuronsand astrocytes. The astrocytes surviving after the toxin application may be stimulated for proliferation and production of growth factorsandgliotransmitterswhichcanimprovestructureand glia-neuronalconnectionsintheprefrontalcortexandrestoreproper behavior.However,thishypothesisneedsfurtherstudies.
Acknowledgments
WewishtothankAssoc.Prof.MarzenaMackowiakforher valu-ablecommentsandassistanceinusingconfocalmicroscopy.
This study was supported by the grant “Depression-mechanisms-therapy”POIG.01.01.02-12-004/09.
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