Prenatal caffeine intake differently affects synaptic proteins during fetal brain development
Artigo publicado no periódico Int. J. Devl Neuroscience 36: 45–52, 2014
Int.J.DevlNeuroscience36(2014)45–52
ContentslistsavailableatScienceDirect
InternationalJournalofDevelopmental Neuroscience
jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / i j d e v n e u
Prenatalcaffeineintake differentlyaffectssynaptic proteinsduring fetalbraindevelopment
SabrinaMioranzza,FernandaNunes,DanielaM.Marques,GabrielaT.Fioreze,
AndréiaS.Rocha,PauloHenriqueS.Botton,MarceloS.Costa,LisianeO.Porciúncula∗
LaboratóriodeEstudossobreoSistemaPurinérgico,ProgramadePós-Graduac¸ãoemCiênciasBiológicas/Bioquímica,PortoAlegre,RS90035-003,Brazil
a r t i c l e i n f o
Articlehistory:
Received12March2014
Receivedinrevisedform23April2014 Accepted30April2014
Keywords:
Caffeine
Braindevelopment BDNF
Shh
Synapticproteins
a b s t r a c t
Caffeineisthepsychostimulantmostconsumedworldwide.However,littleisknownaboutitseffects duringfetalbraindevelopment.Inthisstudy,adultfemaleWistarratsreceivedcaffeineindrinking water(0.1,0.3and1.0g/L)duringtheactivecycleinweekdays,twoweeksbeforematingandthroughout pregnancy.Cerebralcortexandhippocampusfromembryonicstages18or20(E18orE20,respectively) werecollectedforimmunodetectionofthefollowingsynapticproteins:brain-derivedneurotrophicfactor (BDNF),TrkBreceptor,SonicHedgehog(Shh),GrowthAssociatedProtein43(GAP-43)andSynaptosomal- associated Protein25(SNAP-25). Besides,theestimation ofNeuN-stained nuclei(matureneurons) andnon-neuronalnucleiwasverifiedinbothbrainregionsandembryonicperiods.Caffeine(1.0g/L) decreasedthebodyweightofembryosatE20.CorticalBDNFatE18wasdecreasedbycaffeine(1.0g/L), whileitincreasedatE20,withnomajoreffectsonTrkBreceptors.Inthehippocampus,caffeinedecreased TrkBreceptoronlyatE18,withnoeffectsonBDNF.Moderateandhighdosesofcaffeinepromotedan increaseinShhinbothbrainregionsatE18,andinthehippocampusatE20.Caffeine(0.3g/L)decreased GAP-43onlyinthehippocampusatE18.TheNeuN-stainednucleiincreasedinthecortexatE20by lowerdoseandinthehippocampusatE18bymoderatedose.Ourdatarevealedthatcaffeinetransitorily affectsynapticproteinsduringfetalbraindevelopment.TheincreasednumberofNeuN-stainednuclei byprenatalcaffeinesuggestsapossibleaccelerationofthetelencephalonmaturation.Althoughsome modificationsinthesynapticproteinsweretransient,ourdatasuggestthatcaffeineeveninlowerdoses mayalterthefetalbraindevelopment.
©2014ISDN.PublishedbyElsevierLtd.Allrightsreserved.
1. Introduction
Caffeineisoneofthemostconsumedpsychostimulantpresent inourdietindrinkslikecoffee,teaandcolasoftdrinks.Thepharma- cologicalactionsofcaffeineconsistinblockingadenosineactions, anucleosidethatactsasaneuromodulatorinthecentralnervous system(CNS).Adenosinecontrolsneurotransmitterrelease,neu- ronalexcitabilityandcircadianrhythmthroughitsmetabotropic receptorsA1,A2A,A2BandA3(Cunha,2001;Fredholmetal.,2005).
Themajoreffectofcaffeineattheconcentrationsrelevanttodaily intakeistheantagonismofA1andA2Areceptors(Fredholmetal.,
∗Correspondingauthorat:UniversidadeFederaldoRioGrandedoSul,Departa- mentodeBioquímica,InstitutodeCiênciasBásicasdaSaúde,RuaRamiroBarcelos, 2600–anexo,BairroSantana,PortoAlegre,RS90035-003,Brazil.
Tel.:+555133085556;fax:+555133085540.
E-mailaddresses:[email protected],[email protected] (L.O.Porciúncula).
2005).Itiswelldocumentedthebeneficialeffectsofcaffeineon cognitivefunctionsindifferentagesinadultanimals(Angelucci etal.,1999;Costaetal.,2008a;forreviewseeCunhaandAgostinho, 2010).Chroniccaffeinepreventsmemorydisturbancesassociated withaging (Arendash etal., 2006;Costa etal., 2008b;Prediger etal.,2005;Sallaberryetal.,2013)andexperimentalmodelsof Alzheimer’sdisease(Arendashetal.,2009;Dall’Ignaetal.,2007;
Espinosaetal.,2013).
Regarding effects of caffeine during brain development epi- demiological studies have associated caffeine intake and birth outcomes,suchas low birthweight, intrauterinegrowthretar- dation and miscarriage(Brent etal., 2011; Bakker et al.,2010;
Giannellietal.,2003).However,theeffectsofcaffeineintakedur- ingfetalandevenpostnatalbraindevelopmenthavebeenpoorly investigated (for review see Porciúncula et al., 2013; Temple, 2009).Caffeineentersintoalltissuesofthefetus(Brackenetal., 2003)sinceitcaneasilycrossbiologicalmembranesincludingthe placentalbarrierandfetalbrain(Arnaud,1987).Someexperimen- talstudiesinrodentsrevealedthatcaffeineadministeredduring
http://dx.doi.org/10.1016/j.ijdevneu.2014.04.006
0736-5748/©2014ISDN.PublishedbyElsevierLtd.Allrightsreserved.
66
46 S.Mioranzzaetal./Int.J.DevlNeuroscience36(2014)45–52
pregnancyreducedthenumberofsomitesandextentoftheneu- raltubeinthepost-implantationembryo,suggestingintrauterine growth retardation (Jacombs et al., 1999). Besides, caffeine in pregnantfemalesdecreasedtheimmunocontentofmetabotropic glutamatereceptorsofgroupIinthebraininfetusesandpregnant rats(Leónetal.,2005),andalsoinfluencedserotonergicsystem inchickembryo,increasingthecontentsof5-HTanditsmetabo- lite5-hydroxyindoleaceticacid(5-HIAA)(Lietal.,2012).Arecent studyassessingtheeffectofmoderatecaffeineintakeduringpreg- nancy on mice pups resulted in delayed migration of cortical
␥-aminobutyricacid(GABA)neurons,anincreasedsusceptibility toseizures,anddeficitsinspatialmemorywhenassessedinadult- hood(Silvaetal.,2013).
Since caffeine is a non-selective antagonist of A1 and A2A adenosinereceptorsandcouplingofthesereceptorstoG-proteins occurspostnatallyincortexandhippocampus(Adénetal.,2000;
Gaytan et al., 2006), most experimental studies with caffeine duringbrain developmenthave investigated theimpact ofcaf- feineonthesereceptorsatpostnatalstagesorduringadulthood (Adénetal.,2000;GaytanandPasaro,2012;GuilletandKellogg, 1991).However,itremains tobeinvestigatedifthepatternsof thedevelopment of synapses during intrauterine can be mod- ified by caffeine intake. The effects of caffeine administration onproteins, hormones, signalingpathwaysand especially neu- rotrophicfactorsthatregulatethedevelopmentoftheCNSduring intrauterinelifearestilllittleinvestigated.Thus,ourstudyaimed toevaluatetheeffectsofadministrationofdifferentdosesofcaf- feine during pregnancy on proteins involved in the formation andmaturationofsynapses,namely:(i)theneurotrophinbrain- derivedneurotrophicfactor(BDNF)anditsreceptorTrkB;(ii)two componentsofsynapse(axonsandnerveterminals):GrowthAsso- ciatedProtein43 (GAP43)and Synaptosomal-associatedprotein 25(SNAP-25);(iii) themajormorphogenagentSonichedgehog (Shh).Inaddition,anestimation ofthenumberofneuronal and non-neuronalcellswasassessedinthecortexandhippocampusin fetusesfromembryonicstate18and20(E18andE20)exposedto caffeinethroughoutintrauterinelife.
2. Materialsandmethods 2.1. Animalsandcaffeinetreatment
FemaleWistarrats(2monthsold)wereobtainedfromtheCentralAnimalHouse ofourDepartment.Theyweremaintainedinstandardcagesunderastandard dark–lightcycle(lightsonbetween7A.M.and7P.M.),ataroomtemperatureof 22±2◦C.AllexperimentalprocedureswereperformedaccordingtotheEthicalcom- mitteeofUniversidadeFederaldoRioGrandedoSul(Proc.N◦20332)incompliance withBrazilianSocietyforNeuroscienceandBehavior(SBNeC)recommendationsfor animalcare.
AdultfemaleWistarratsweretreatedwithcaffeineindrinkingwater(0.1, 0.3and1.0g/L,correspondingtolow,moderateandhighintake,respectively) (Fredholmetal.,1999),duringtheactiveperiod(darkcycle)inweekdays,two weeksbeforemating.Although1mg/mLofcaffeineisconsideredhighconsump- tionofcaffeine,ityieldedaplasmaconcentrationoflessthan60Minadultrats (Costenlaetal.,2010;Duarteetal.,2009).Atthebeginningoflightcycleallexper- imentalgroupsreceivedtapwater.Thepresenceofvaginalplugwasconsidered asembryonicday0(E0).Thesamecaffeinetreatmentcontinuedduringpregnancy uptoembryonicdays18or20(E18orE20,respectively),whenthedamswere sacrificedunderanesthesia.Thefetuseswereweighedandwholecortexandhip- pocampuswereimmediatelydissectedoutat4◦CforWesternBlotanalysis;orthe wholebrainwereimmersedin4%paraformaldehydein0.1MPBSforfixationof thebraintissuetodeterminetotalnumberofneuronalandnon-neuronalcellsin cortexandhippocampusbytheisotropicfractionator.Thelittersizeandnumberof implantationswerealsoanalyzed.
Themoderatedoseof0.3g/Lcaffeinewaschosenconsideringtheplasmacon- centrationofcaffeineinratdamsbeingthisdosesimilartothatfoundinbloodof humanmothersdrinkingthreetofourcupsofcoffeeperday(Adénetal.,2000).The othertwoconcentrationswerechosentoreachapproximately3timeslowerand3 timeshigherconcentrationscomparingwiththemoderatedose(0.1g/Land1.0g/L, respectively),inordertohaveawiderangeofcaffeineintake.Femaleratsreceived caffeinesolutionsonlyduringthedarkcycle,whichistheiractiveperiod,tomimic thepatternofcaffeineconsumptioninhumans.Duringthelightcycle,caffeine
solutionswerereplacedbywateradlibitum.Allexperimentalgroupsconsumedsim- ilaramountsofcaffeinated-wateranddrinkingwater.Althoughwedidnotquantify theplasmalevelsofcaffeine,thechosentreatmentregimensarethoughttocorre- spondtoalow,moderateandhighcaffeineintakeinhumans,witheffectsbelieved tobemainlyoperatedthroughantagonismofadenosinereceptors(Fredholmetal., 1999).
2.2. SDS-PAGE(sodiumdodecylsulfate-polyacrilamide)immunoblotting
HippocampusandwholecortexweredissectedoutfromE18orE20(pooloftwo fetusesperlitter,consideringonelitterasonesubject),wereimmediatelyhomog- enizedin5%SDSwithaproteaseandphosphataseinhibitorcocktail(Sigma,São Paulo/Brazil)andfrozenat−70◦C.Afterdefrost,theproteincontentwasdetermined byBicinchoninicacidassayusingbovineserumalbumin(BSA)asstandard(Pierce, SãoPaulo/Brazil).Sampleextractsweredilutedtoafinalproteinconcentrationof 2g/LinSDS-PAGEbuffer.Aliquotscorrespondingto20–40gproteinforhip- pocampusandwholecortexsampleswereseparatedbySDS-PAGE(12%runninggel witha4%concentratinggel)andelectro-transferredtonitrocellulosemembranes.
AfterblockingwithTris-bufferedsaline0.1%Tween-20(TBS-T)containing3%bovine serumalbuminfor1h,themembraneswereincubatedfor24hoursat4◦Cwiththe followingprimaryantibodies:mouseanti-BDNF(1:500,Sigma,SãoPaulo/Brazil);
rabbitanti-TrkB(1:1000,Abcam,SãoPaulo/Brasil);rabbitanti-GAP-43(1:2000, Sigma,SãoPaulo/Brazil);mouseanti-Shh(1:500, Abcam,SãoPaulo/Brazil);or rabbit anti-SNAP-25 (1:1000, Sigma, São Paulo/Brazil). The membranes were then washed with TBS-T, incubatedwith horseradish peroxidase conjugated secondaryantibodyfor1hatroomtemperatureanddevelopedwithchemilu- minescenceECLkit(Amersham,SãoPaulo/Brazil).Membraneswerere-probed for-tubulinimmunoreactivity.Theautoradiographicfilmswerescannedand densitometricanalyseswereperformedusingpublicdomainNIHImageProgram (developedattheU.S.NationalInstitutesofHealthandavailableontheinternetat http://rsb.info.nih.gov/nih-image/).Bandintensitywasnormalizedto-tubulinas aloadingcontroltoassessproteinlevels.Thedatausedinstatisticalanalysiswere obtainedfromtheratiooftheproteinstudiedand-tubulindensityunitlines.
2.3. Quantificationofneuronalandnon-neuronalnuclei
Weusedtheisotropicfractionatormethodtoestimatetotalnumberofcellsand neurons,aspreviouslydescribed(Herculano-HouzelandLent,2005).Wholecortex andhippocampusfromthefixedbrainwereweighedandmechanicallydissociated inasalinesolutionwith0.1%TritonX-100andturnedintoanisotropicsuspension ofisolatednuclei.Thetotalnumberofnucleiinsuspension–andthereforethe totalnumberofcellsintheoriginaltissue–wasestimatedbydeterminingthe densityofnucleiinsmallaliquotsstainedwiththefluorescentDNAmarkerDAPI (4,6-diamidino-2-phenylindoledihydrochloride),underthemicroscopewitha40×
objective,usingahemocytometerforquantification.Neuronalnucleifromanaliquot ofthesuspensionwereselectivelyimmunolabeledovernight,atroomtemperature, withmousemonoclonalanti-NeuNantibody(Millipore,MAB377)atadilutionof 1:200inPBS.AfterwashingthenucleiinPBS,theywereincubatedfor2hatroom temperaturewithAlexaFluor488anti-mouseIgGsecondaryantibody(Invitrogen, BiogenSãoPaulo/Brazil),atadilutionof1:500inPBSinthepresenceof10%normal goatserum.TheneuronalfractionineachsamplewasestimatedbycountingNeuN- labelednucleiinatleast500DAPI-stainednuclei.Imageanalysisandquantification weremanuallyperformedandalsoconfirmedbyusingsoftwarefromNikonEclipse E600.Theresultswerepresentedasnumberofcellspermgtissue.
2.4. Statisticalanalysis
Statistical analysis was performed by using One-way ANOVA and Newman–Keulsposthoctest.ThestatisticalsignificancewasconsideredforP<0.05.
3. Results
3.1. Reproductivedata
The litter size and the number of implantations were not affectedbychroniccaffeine,asshowninTable1.Thefetalweight wasdecreasedby16%inembryos(E20)fromdamsthatreceived caffeineatthehighestdose(1.0g/L)(Table1).
3.2. Theimpactofdifferentcaffeinedosesduringpregnancyon BDNFsignalingandonimportantproteinsforsynaptic development
Prenatalcaffeineintakedifferentlyaffectedproteinscrucialto fetalbraindevelopmentintwodistinctembryonicstagesinwhole cortexand hippocampus. AtE18 inhippocampus, highcaffeine
67
S.Mioranzzaetal./Int.J.DevlNeuroscience36(2014)45–52 47
Table1
Reproductivedataa
Water Caffeine0.1g/L Caffeine0.3g/L Caffeine1.0g/L
No.ofdams
Embryonicday18 7 7 7 7
Embryonicday20 7 7 7 7
No.ofimplantations
Embryonicday18 10.44±0.48 10.36±0.61 10.73±0.78 10.67±0.44
Embryonicday20 10.00±0.52 10.25±0.48 10.50±0.72 10.09±0.86
Meanfetalweight(g)
Embryonicday18 1.47±0.09 1.47±0.01 1.41±0.05 1.41±0.04
Embryonicday20 3.88±0.13 3.84±0.16 3.71±0.07 3.26±0.13b
aDataarepresentedasmean±S.E.M.
bDifferentfromcontrolgroup(P<0.05).
intake(1.0g/L)decreasedTrkB(Fig.1A,blacksquare)andincreased Shh(Fig.2A),whereas themoderate dosereducedhippocampal GAP-43(Fig.3A);in wholecortexBDNFwasdecreased(Fig.1C, blackcircle)and Shhwasincreased(Fig.2B)infetuses exposed tothehighestdoseofcaffeine.Whenassessingembryonicstage 20 (E20), BDNFinwholecortex wasincreased(Fig.1D)bythe
highestdose,andboth moderateandhighcaffeinedose(0.3g/L and1.0g/L,respectively)increasedShh(Fig.2A)inhippocampusat E20.TheimmunocontentofSNAP-25wasnotaffectedbycaffeine intake(Fig.4AandB).Thelowestdoseofcaffeinewasdevoidof effectinallproteinsassessedinwholecortexandhippocampusfor bothembryonicstages.
Fig.1.ImmunoblottinganalysisofBDNF(blackcircle)anditsreceptor,TrkB(blacksquare)inthehippocampusatE18(A)andE20(B),andinthewholecortexatE18(C) andE20(D)fromfetusesexposedtowaterorcaffeine.Dataaremeans±S.E.M(n=5–6samples/group)ofdensityunitlinesobtainedfromprotein/tubulinratio.*P<0.05, significantlydifferentfromcontrolgroup(water).One-wayANOVAfollowedbyNewman–Keulsmultiplecomparisontest.
68
48 S.Mioranzzaetal./Int.J.DevlNeuroscience36(2014)45–52
Fig.2.ImmunoblottinganalysisofShhinthehippocampus(A)atE18(blacksquare)andE20(whitecircle),andinthewholecortex(B)atE18(blacksquare)andE20(white circle)fromfetusesexposedtowaterorcaffeine.Dataaremeans±S.E.M(n=5–6samples/group)ofdensityunitlinesobtainedfromShh/tubulinratio.*P<0.05,significantly differentfromcontrolgroup(water).One-wayANOVAfollowedbyNewman–Keulsmultiplecomparisontest.
3.3. Numberofneuronalandnon-neuronalcellsisaffectedby caffeineintakeincortexandhippocampusatdifferentembryonic stages
Theimpactofprenatalcaffeineintakeatdifferentdoseswasalso evaluatedinnumberofneuronalandnon-neuronalcells.Caffeine 0.3g/Lincreasedtotalnumberofcellsandneuronalcellsinhip- pocampusfromfetusesexposedtocaffeineuptoE18(Fig.5A).The lowestdoseofcaffeineincreasedneuronalcellsandtotalnumber ofcellsincortexatE20(Fig.5D).Wedidnotfoundanyeffectofcaf- feinetreatmentinhippocampusatE20orincortexatE18(Fig.5B andC,respectively).
4. Discussion
Inthisstudy,theimmunocontentofkeyproteinsinsynapticfor- mationandthetotalnumberofneuronalandnon-neuronalcells wereassessedinwholecortexandhippocampusfromratembryos
exposedtodifferentdosesofcaffeine.Translatingtohumanbrain development,thelimbicsystemandcortexfromratembryosat E18correspondtothefirsttrimesterofpregnancyinhumans,while thesebrainareasatE20correspondtotheendoffirstandthebegin- ningofsecondtrimester (Clancyet al.,2001).These embryonic stagescomprisetheperiodofhighneuronal differentiationrate atE18forwholecortex,whiletheneuronalmigrationandlaminar organizationoccuratE20(Naveauetal.,2014).Inthehippocampus duringtheprenatalperiod,neuronsfortheAmmon’shornaregen- erated,whilethemajorityofthosedestinedforthedentategyrus areformedpostnatally(Bayeretal.,1993).
Inagreementwithpreviousstudies,albeitwithdifferentsched- ules of administration, theabsenceof effect onthe number of implantationsandlittersizewasalsoobservedinanimalstreated withcaffeine(Everekliogluetal.,2003;Lorenzoetal.,2010).Oneof theexpectedeffectsofhighdoseofcaffeineisthedecreaseinthe bodyweight,whichwasconfirmedatE20.Thisphenomenonwas previouslyreportedforpregnantWistarratsthatreceivedcaffeine
Fig.3. ImmunoblottinganalysisofGAP-43inthehippocampus(A)atE18(blacksquare)andE20(whitecircle),andinthewholecortex(B)atE18(blacksquare)andE20 (whitecircle)fromfetusesexposedtowaterorcaffeine.Dataaremeans±S.E.M(n=5–6samples/group)ofdensityunitlinesobtainedfromGAP-43/tubulinratio.*P<0.05, significantlydifferentfromcontrolgroup(water).
69
S.Mioranzzaetal./Int.J.DevlNeuroscience36(2014)45–52 49
Fig.4.ImmunoblottinganalysisofSNAP-25inthehippocampus(A)atE18(blacksquare)andE20(whitecircle),andinthewholecortex(B)atE18(blacksquare)and E20(whitecircle)fromfetusesexposedtowaterorcaffeine.Dataaremeans±S.E.M(n=5–6samples/group)ofdensityunitlinesobtainedfromSNAP-25/tubulinratio.No statisticaldifferenceswerefound.
Fig.5.NumberofNeuN-stainednuclei(estimatedasneuronalcells)andDapi-stainednuclei(estimatedasnon-neuronalcells)(permgtissue)inthehippocampusatE18 (A)orE20(B),andinthewholecortexatE18(C)andE20(D)fromfetusesexposedtowaterorcaffeine.*P<0.05,significantlydifferentfromcontrolgroup(water).One-way ANOVAfollowedbyNewman–Keulsmultiplecomparisontest.
70
50 S.Mioranzzaetal./Int.J.DevlNeuroscience36(2014)45–52
intragastricallyattheconcentrations20,60, and180mg/kg/day fromgestationalday11(GD11)toGD20.Inthatstudy,alltreat- mentsreducedfetalbodyweightatembryonicday20(E20)and therewasanincreaseinglucocorticoidreceptorinthefetalhip- pocampus(Xuetal.,2012).Althoughitisdifficulttodiscriminatea moredirecteffectofcaffeinefromthelossofbodyweight,themod- ificationsfoundinsynapticproteinswerenotrestrictedtoembryos atE20.
The analysis of some proteins involved in the assembly of thesynapseswasinvestigatedintwoimportantbrainareasfrom embryosthat receivedcaffeine.The brain-derivedneurotrophic factor(BDNF)isoneofthemainproteinsinvolvedintheregulation, structureandmaintenanceofsynapsesduringbraindevelopment andalsoinadulthood(LuandChow,1999;Poo,2001;Tylerand Pozzo-Miller,2003).SignalingbyBDNFalsoparticipatesasaregula- torofsynapticretrogradedevelopmentandisessentialforgrowth andelongationofdendritesofexcitatoryandinhibitorysynapses (Carvalhoetal.,2008;LipskyandMarini,2007;Singhetal.,2006).
ItiswellknownthatBDNFsignalingthroughitsreceptorTrkBcan influencethemorphologyandsynapticconnectivityofdeveloping neurons(HuangandReichardt,2001;Poo,2001).
Shh is a major morphogen agent, and is thought to be a keyregulator of neuraland oligodendroglialprogenitors across development (Desouza et al., 2011). Possible alterations in nerve terminals were also assessed by the immunocontent of synaptosomal-associatedprotein25(SNAP-25),whichisaprotein componentofthesolubleNSF(N-ethylmaleimide-sensitivefactor) attachmentproteinreceptor(SNARE)complex;theincreasedlevels ofSNAP-25arerelatedtosynaptogenesisandmaturationoffunc- tionalsynapticconnectivityduringbrain development(Catsicas etal.,1991).Wealsoassessedtheimpactofcaffeineintakedur- ingpregnancyinGrowthAssociatedProtein43(GAP-43),another protein component of axons and nerve terminals (reviewed in BenowitzandRouttenberg,1997;Oestreicheretal.,1997),whichis associatedwithneuriteoutgrowthandsynapseformationinbrain (Baetgeetal.,1992;McGuireetal.,1988).
Weinvestigatedtheimpactofdifferentdosesofcaffeineinpro- teinsrelatedtofetalhippocampaldevelopment.AtE18,moderate andhighcaffeineintakedifferentlyaffectedTrkB,GAP-43andShh.
OneoftheconsequencesoflackingexpressionofGAP-43istheinhi- bitionofproliferationofcertainneuralprecursorsanddisturbances inthematurationofdifferentiated neurons(Maier etal.,1999).
Thisproteiniscrucialforaxonalguidance,sinceGAP-43knockout miceshowdisrupted corticalmapsand absenceof corticalbar- rels(Maieretal.,1999).Besides,anincreaseinGAP-43expression potentiatedneuriteoutgrowthinculturedhippocampalneurons (Korshunovaetal.,2007).Wesupposedthatpossibledeleterious effectsbycaffeine-induceddecreaseofGAP-43mighthavebeen offsetbythegradualincreaseinthehippocampalShh.Ofnote,the changesobservedatE18intheTrkBandGAP-43immunocontent werenolongerobservedatE20.
SinceaddingShhinculturedhippocampalneuronsbecomethe presynapticterminalssignificantlylarger,withvariableincreases inthesizeofsynapticvesicles(Mitchelletal.,2012),weassessed SNAP-25thatcanbeeasilyimmunodetectedatearlystagesofbrain development(PrescottandChamberlain,2011).Notably,caffeine inalldosesadministereddidnotmodifySNAP-25inbothagesand brainregions,suggestingthatatthisstageofbraindevelopment therewasnoimpactofthispsychostimulantonnerveterminals.
Differentlyfromthe hippocampus, higher caffeineincreased corticalBDNFatE18anddecreased thisproteinatE20,withno effectonTrkBreceptors.Coincidently,Shhwasincreasedinthecor- texfromfetusesatE18,butreturnedtonormallevelsatE20.One oftheconsequencesofincreasedBDNFandShhcausedbycaffeine maybeanaccelerationoftelencephalondevelopment,anevent previouslyreportedininvitrostudies(Marretetal.,1997;Sahir
etal.,2000,2001).Shhmodulatescellproliferationandthemigra- tionofprogenitorcellsinadultbrain(Charytoniuketal.,2002;Dave etal.,2011;Palmaetal.,2005;Traiffortetal.,2001).Thismor- phogenishighlyexpressedinratbrainsfromE12topostnatalday2 (P2)(Petraliaetal.,2011),andrecentlyitsexpressionhasbeeniden- tifiedincorticalpyramidalneurons(Garciaetal.,2010).Ourresults arealsoinlinewithinvitrostudiesinwhichcaffeineatrelatively highconcentrationspromotedanoverexpressionofShhcultured neuronsand astrocytes (Sahir et al.,2004).Importantly, Shh is relatedtoinductionofGABAergicinterneurondifferentiationin telencephalonatearlyembryonicstages(E11.5)(MadyandKohtz, 2007).Oneoftheconsequencesofincreasingthismorphogeninlate pregnancymightbeanabnormaldistributionofneuronsincorti- callayersbeforebirth,whichmayimpactGABAneuronmigration postnatally.Interestingly,moderatecaffeineintake(0.3g/L)during pregnancyandlactationdelayedGABAneuronmigrationinthehip- pocampusandsuperficialcorticallayersatpostnatalday6(PND6) (Silvaetal.,2013).
Theparticipationofotherproteinsandsignalingcascadesin theincreaseofNeuN-stained nucleiinthecortexbylower caf- feinedeservesmoreinvestigation.Giventhecomplexityofcerebral cortex compared to hippocampus, it is not possible to infer if thisincreasecouldreflectchangespostnatallyespeciallybecause otherdevelopmentalmilestoneswerenotassessed.However,itis importanttonotethatnoeffectwasfoundforlowercaffeinein theneurotrophinsandtheotherproteinsinvolvedinthesynaptic developmentsuchasShhpathway,whichcoordinatesdifferentia- tionofneuralstemcellsintheneocortex.Infact,theupregulation ofShhsignalingwasdescribedtopromotetheincreaseofthickness andsizeoftheneocortex(Shikataetal.,2010).
Fromourknowledge,thisisthefirststudyreportingeffectsof prenatalcaffeineinthenumberofNeuN-stainednuclei(asesti- mationofthenumberofmatureneurons).Theimpactofchronic caffeine hasalready been investigated in the adultneurogene- sis,withdifferentfindings.Forexample,chronicadministrationof caffeinesuppressedhippocampalsubgranularzone(SGZ)cellpro- liferationandneurogenesisinrats(Hanetal.,2007;Kochmanetal., 2009).Ontheotherhand,inamoredetailedstudy,theeffectsofcaf- feineonproliferationofneuronalprecursorsweredosedependent;
moderatedosesdepressingwhilehigherdosesincreasingprolifer- ation(WentzandMagavi,2009).Inthesamestudy,thesurvival anddifferentiationof neuronsfollowingcaffeineadministration wasnotaffected,whichcontrastswithdatafromHanetal.(2007).
Here,theincreasednumberofNeuN-stainednucleibycaffeinewas alsodependentofthedoseandspecificaccordingtobrainregion andembryonicperiod.Itisimportanttotakeintoaccountthatthe fetalmetabolismofcaffeineisnotfullymature,andcertainlyits effectsaremorepronouncedgiventheprolonghalf-lifeinthefetus (Arnaud,1987,2011).
Overall,ourdatashowthatcaffeineexposurethroughoutthe periodofprenatalbraindevelopmentcanproducesignificant,but transitoryeffectsonfetalbraindevelopment.Itisimportanttonote thatsnapshotsofeventsduringbraindevelopmentmayyieldpar- tialandsometimesconflictinginformation.Thedatapresentedalso highlighttherelevanceofcaffeineintakeonimportantsynaptic proteins.Untilnow,thevastmajorityofmoststudiesfocusedon theeffectsofcaffeineinthematurebrain.Certainly,theimpactof caffeineintakeonsynapseremodelingormodulationofsynaptic efficacyandplasticityintheimmaturebraindeservesmoreatten- tion.
Acknowledgments
ThisresearchwassupportedbyCAPES,FAPERGS(PRONEXand PRONEM),andCNPq(INCT).
71