Gabaergic and opioid receptors mediate the facilitation of NaCl intake induced by alfa 2-adrenergic activation in the lateral parabrachial nucleus.

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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

Gabaergic

and

opioid

receptors

mediate

the

facilitation

of

NaCl

intake

induced

by

␣

2

-adrenergic

activation

in

the

lateral

parabrachial

nucleus

C.A.F.

Andrade

a,∗

_,

_L.B.

_De

_Oliveira

b

_,

_G.M.F.

_{Andrade-Franzé}

a

_,

_L.A.

_De

_Luca

_Jr

a

_,

Débora

S.A.

Colombari

a

_,

_J.V.

_Menani

a

a_Departament_of_Physiology_and_Pathology,_School_of_Dentistry,_UNESP,_{Araraquara-SP,}_14801-903,_Brazil b_Department_of_Biological_Sciences,_DECBI-NUPEB,_Federal_University_of_Ouro_Preto,_Ouro_Preto,_MG,_Brazil

h

i

g

h

l

i

g

h

t

s

•␣2-adrenoceptoractivationintheLPBNincreasessodiumintakeinﬂuid-depletedrat.

•Opioidergicreceptorblockadepartiallyreduced␣2-adrenoceptoractivationeffects.

•␣2-adrenoceptoractivationeffectsarepartiallyreducedbyGABAAreceptorblockade.

•Opioidergic/GABAergicblockadepartiallyreduced␣2-adrenoceptoractivationeffects.

•␣2-adrenoceptoractivationeffectsarepartiallydependentonopioid/GABAAreceptors.

a

r

t

i

c

l

e

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n

f

o

Articlehistory:

Received14July2014

Receivedinrevisedform1October2014 Accepted6October2014

Availableonline14October2014 Keywords: Sodiumappetite Adrenergic GABA Opioid Dehydration Thirst

a

b

s

t

r

a

c

t

Alpha2-adrenergic, gabaergic or opioidergic activation in the lateral parabrachial nucleus (LPBN) increasessodiumintake.Inthepresentstudy,weinvestigatedtheeffectsofsingleorcombined block-adeofopioidergicandgabaergicreceptorsintheLPBNontheincreaseof0.3MNaClintakeinducedby ␣2-adrenoceptoractivationintheLPBN.MaleHoltzmanrats(n=5–9/group)withcannulasimplanted bilaterallyintheLPBNweretreatedwiththediureticfurosemide(10mg/kgbwt.)combinedwithlow doseoftheangiotensinconvertingenzymeinhibitorcaptopril(5mg/kgbwt.)subcutaneously. Bilat-eralinjectionsofmoxonidine(alpha2-adrenergic/imidazolinereceptoragonist,0.5nmol)intotheLPBN increasedfurosemide+captopril-induced0.3MNaClintake(25.8±1.4,vs.vehicle:3.8±1.1ml/60min). Theopioidergicreceptorantagonistnaloxone(100nmol)ortheGABAAreceptorantagonistbicuculline (5nmol) injected intothe LPBN partially reducedthe increaseof 0.3M NaCl intake produced by LPBNmoxonidine(11.8±4.0and22.8±4.5,respectively,vs.vehicle+moxonidine:31.6±4.0ml/60min, respectively).Similartothetreatmentwitheachantagonistalone,thecombinedinjectionsof nalox-one(100nmol)andbicuculline(5nmol)intotheLPBNalsopartiallyreducedmoxonidineeffectson 0.3MNaClintake(15.5±6.5ml/60min).TheGABABreceptorantagonistsaclofen(5nmol)injectedinto theLPBNdidnotchangetheeffectsofmoxonidineon0.3MNaClintake(24.3±7.8ml/120min).These resultssuggestthattheincreaseof0.3MNaClintakeby␣2-adrenergicreceptoractivationintheLPBNis partiallydependentonGABAAandopioidreceptoractivationinthisarea.

1. Introduction

The lateral parabrachial nucleus (LPBN) is a pontine area stronglyinvolvedwithinhibitorymechanismsthatcontrolwater

∗ Correspondingauthor.Address:RuaHumaitá1680,Araraquara–SP,14801-903, Brasil,Tel.:+551633016487;fax:+551633016488.

E-mailaddress:carina.andrade@foar.unesp.br(C.A.F.Andrade).

and NaCl intake [1–7]. The LPBN is reciprocally connected to forebrain areas implicated in the maintenance of blood pres-sure and body ﬂuid homeostasis, such as the paraventricular nucleus ofthehypothalamus, thecentralnucleus of the amyg-dala and the median preoptic nucleus. The LPBN is also richly interconnectedwithmedullaryregions,which includesthearea postrema(AP)andthemedialportionofthenucleusofthe soli-tarytract(mNTS),[8–15].Therefore,theLPBNreceivestasteand visceralsignalsthatascendfromAP/mNTSenroutetoforebrain

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intothe LPBN strongly increases 0.3MNaCl intake by satiated orsodiumdepleted rats[20,27,29,30].Therefore, theactivation ofGABAA,GABAB,opioid receptorsin theLPBNdeactivatesthe

inhibitorymechanisms,releasingsodiumintakeifexcitatory sig-nalswereactivatedbysodiumdepletionornot[20,27,29].

Itisnot knownif␣2-adrenoceptor,GABAergicand

opioider-gicmechanismsinteractintheLPBNtosopotentlyinducesodium intake.In thepresentstudy,we investigatedtheeffectof com-binedantagonismofopioidergic and/orgabaergicreceptorwith ␣2-adrenoceptoractivationintheLPBNon0.3MNaClandwater

intakeinducedbyFURO+CAPinrats.

2. Materialandmethods

2.1. Animals

MaleHoltzmanratsweighing290to310gwereused.The ani-malswerehousedinindividualstainlesssteelcageswithfreeaccess tonormalsodiumdiet(GuabiRatChow,Paulinia,SP,Brazil),water and0.3MNaClsolution.Temperaturewasmaintainedat23±2◦C, andhumiditywasmaintainedat55±10%ona12:12light-dark cyclewithlightonsetat7:30AM.

Theexperimental protocolsused in the present study were approvedbyEthicalCommitteeforAnimalCareandUsefrom Den-tistrySchoolofAraraquara–UNESP,Brazil(protocolno:06/2006) andfollowedtherecommendationsfromtheNationalCouncilfor theControlofAnimalExperimentation(CONCEA)andtheAmerican NationalInstituteofHealthGuidefortheCareandUseofLaboratory Animals(NIHpublicationsNo.80–23,1996,USA).Alleffortswere madetominimizeanimaldiscomfortandthenumberofanimals used.

2.2. Cerebralcannulas

Rats were anesthetized with ketamine (80mg/kg of body weight) combinedwith xylazine(7mg/kg of body weight) and placed in a Kopf stereotaxic instrument. The skull was leveled betweenbregmaandlambda.Bilateralstainlesssteel23-gauge can-nulaswereimplantedindirectiontotheLPBNusingthefollowing coordinates:9.4mmcaudaltobregma,2.1mmlateraltothe mid-line,and4.2mmbelowtheduramater.Thetipsofthecannulas werepositionedatapoint2mmaboveeachLPBN.Thecannulas wereﬁxedtothecraniumusingdentalacrylicresinandjeweller screws.A30-gaugemetalobturatorﬁlledthecannulasbetween tests.Theratswereallowedtorecover6daysbeforedruginjections intotheLPBN.

donationofSolvayPharma,Hannover,Germany)wasadministered intotheLPBNatthedoseof0.5nmol/0.2␮l[19,21,24,32]. Bicu-culline,(GABAAreceptorantagonist,Tocris,Ellisville,MO,USA)was

administeredintotheLPBNatthedosesof1.6and5.0nmol/0.2_␮l

[27]; saclofen(GABABreceptor antagonist,Tocris, Ellisville,MO,

USA)wasadministeredintotheLPBNatthedoseof5.0nmol/0.2␮l

[30]andnaloxonehydrochloride(opioidergicreceptorantagonist, SigmaChemicals, St.Louis,MO,USA)wasadministeredintothe LPBNatthedoseof100nmol/0.2␮l)[20].

Moxonidine,naloxone,saclofenandbicucullineweredissolved in a mix of propylene glycol/water 2:1 (vehicle). Vehicle was injected as control into the LPBN. Captopril was dissolved in isotonicsaline. Furosemidewasdissolvedinalkalinesaline(pH adjustedto9.0withNaOH).

2.5. Ingestivetest

Ratsweretestedintheirhomecages.Water,0.3MNaCland foodwereremovedandtheanimalsreceivedsubcutaneousFURO (10mg/kg ofbody wt)+CAP(5mg/kg of body wt)as described previously[7,21,31].Onehourlater,waterand0.3MNaClwere providedinburetteswith0.1-mldivisionsthatwereﬁttedwith metaldrinkingspouts.Cumulativewaterand0.3MNaClintakes weremeasuredat15,30,60,90,and120min(ingestivetest).The injectionsofmoxonidineorvehicleintotheLPBNwereperformed 45minafterFURO+CAPtreatmentor15minbeforetheratshad accesstowaterand0.3MNaCl.Theopioidergicand/orGABAergic antagonist,orvehicle,wasinjectedintotheLPBN15minbeforethe injectionofmoxonidineoritsvehicle.

Eachgroupofratswassubmittedtofourtests,eachtestina dif-ferentday,ata48-hourminimuminterval.Ineachtestthegroup wasdividedintwoandeachhalfreceivedadifferenttreatment intotheLPBN.ThesequenceofthetreatmentsintotheLPBNinthe differenttestswasrandomized.Allanimalsreceivedatotaloffour treatmentsintotheLPBN:vehicle+vehicle;vehicle+moxonidine; opioidergicand/orGABAergicantagonist+moxonidine; opioider-gicand/orGABAergicantagonist+vehicle.

2.6. Histology

The animals received bilateral injections of 2% Evans blue solution(0.2_␮l)intotheLPBNafterthefourthingestivetest.They werethendeeplyanesthetizedwithsodiumthiopental(80mg/kg ofbodyweight)andperfusedtranscardiallywithsalinefollowed by10%formalin.Thebrainswereremoved,ﬁxedin10%formalin, frozen, cut in 50-␮m sections, stained with cresyl violet, and analyzedbylightmicroscopytoconﬁrmtheinjectionsitesinto theLPBN.

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Fig.1. Photomicrographofacoronalsectionofaratbrainshowing(arrows)the sitesofbilateralinjectionsintotheLPBN.scp:superiorcerebellarpeduncle.

2.7. Statisticalanalysis

Theresultsarereportedasmeans±S.E.M.Two-way repeated-measures ANOVA(treatment and time as factors) and Student Newman-Keulstestswereusedforcomparison.Differenceswere consideredsigniﬁcantatP<0.05.

3. Results

3.1. Histologicalanalysis

Fig.1showsthetypicalsitesofbilateralinjectionsintotheLPBN. TheLPBNinjectionswerecenteredmainlyinthecentrallateral ordorsallateralportionsoftheLPBN[33].Injectionscenteredin theventrallateralandexternallateralportions,aswellasinthe Kölliker-Fusenucleusobservedinsomeratswerealsoconsidered ascorrectlyplacedintheLPBN.Thesitesoftheinjectionsinthe presentstudyweresimilartothoseofpreviousstudiesthatshowed theeffectsofmoxonidine,muscimoloropioidsinjectedintothe LPBNonNaClandwaterintake[19–21,24,27].

3.2. GABAAreceptorblockadeandmoxonidineinjectedintoLPBN

Bilateral injections of moxonidine (0.5nmol/0.2␮l) into the LPBN strongly increasedFURO+CAP-induced 0.3M NaClintake whencomparedtocontrol(vehicle+vehicle)injections.Bicuculline (5nmol/0.2␮l)partiallyreducedtheincreaseof0.3MNaClinduced bymoxonidineinFURO+CAP-treatedratsduringthewhole inges-tivetest.Bicuculline(5.0nmol/0.2␮l)combinedwithvehicledid notaffectFURO+CAP-induced0.3MNaClintake(Fig.2A).ANOVA showedsigniﬁcanteffectfortreatmentonNaCl0.3Mintake[F(3, 24)=22.9;P<0.05],(Fig.2A).

Moxonidine increased water intake at 120min of the test (Fig. 2B). Bicuculline (5.0nmol) reversed the increase of water intakeinducedbymoxonidineinFURO+CAP-treatedrats. Bicu-culline(5.0nmol)+moxonidine also reduced water intake until 30minofthetestwhencomparedtocontrol(veh+veh)(Fig.2B). Bicuculline(5.0nmol/0.2␮l)combinedwithvehicledidnotaffect FURO+CAP-induced waterintake(Fig.2B).ANOVAshowed sig-niﬁcantinteractionbetweentreatmentandtime[F(12,96)=7.9; P<0.05]forwaterintake(Fig.2B).

Inanothergroupofrats,(Table1)theincreasein0.3MNaCl intakeinducedbymoxonidineinFURO+CAP-treatedratswas par-tiallyreducedbybicuculline(1.6nmol/0.2␮l)until90minofthe

Fig.2.(A)Cumulative0.3MNaClintake;(B)cumulativewaterintakeinducedby s.c.FURO+CAPtreatmentinratsthatreceivedbilateralinjectionsofbicuculline (bicu,5.0nmol/0.2␮l)orvehicle(veh,0.2␮l)combinedwithmoxonidine(moxo, 0.5nmol/0.2␮l)orvehicle(veh,0.2␮l)intotheLPBN.Resultsareexpressedas means±SEM;n=numberofrats.

test.FURO+CAP-inducedwaterintakewasnotmodiﬁedby injec-tionsofmoxonidinealoneorcombinedwithbicuculline(Table1). ANOVA showed signiﬁcant effect for treatment on 0.3M NaCl intake[F(3,12)=26.7;P<0.001],butnoeffectonwaterintake[F(3, 12)=0.8;P>0.05](Table1).

3.3. GABABreceptorblockadeandmoxonidineinjectedintoLPBN

Saclofen (5.0nmol/0.2␮l) did not signiﬁcantly change the increase of FURO+CAP-induced0.3MNaCl intakeproduced by moxonidine(0.5nmol/0.2␮l).Saclofencombinedwithvehicledid notchangeFURO+CAP-induced0.3MNaClintake.ANOVAshowed signiﬁcanteffectfortreatmenton0.3MNaClintake[F(3,18)=9.7; P<0.05](Fig.3A).

FURO+CAP-inducedwaterintakewasnotmodiﬁed by mox-onidine alone or combined with saclofen. ANOVA showed no signiﬁcanteffectfortreatment[F(3,18)=3.1;P>0.05](Fig.3B).

3.4. OpioidreceptorblockadeandmoxonidineinjectedintoLPBN

Naloxone (100nmol/0.2␮l) partially reduced the increase of FURO+CAP-induced 0.3M NaCl intake produced by mox-onidine (0.5nmol/0.2␮l) as indicated by the signiﬁcant differ-ence between treatments [F(3, 12)=10.1; P<0.05] (Fig. 4A).

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combinedwithnaloxone[F(3,12)=1.3;P>0.05],(Fig.4B).

3.5. CombinedopioidandGABAAreceptorblockadeand

moxonidineinjectedintoLPBN

The previous injections of naloxone (100nmol/0.2 ␮l)+bicuculline (5nmol/0.2␮l) into the LPBN, similar to these

Fig.3.(A)Cumulative0.3MNaClintake;(B)cumulativewaterintakeinduced bys.c.FURO+CAPtreatmentinratsthatreceivedbilateralinjectionsofsaclofen (saclo,5.0nmol/0.2␮l)orvehicle(veh,0.2␮l)combinedwithmoxonidine(moxo, 0.5nmol/0.2␮l)orvehicle(veh,0.2␮l)intotheLPBN.Resultsareexpressedas means±SEM;n=numberofrats.

the signiﬁcant differences between treatments for 0.3M NaCl [F(3,12)=16.1;P<0.001]andwaterintake[F(3,12)=9.1;P<0.05] (Fig.5).Theinjectionsofnaloxone(100nmol/0.2␮l)+bicuculline (5nmol/0.2␮l)combinedwithvehicledidnotaffectFURO+ CAP-inducedwaterand0.3MNaClintake(Fig.5).

Fig.4. (A)Cumulative0.3MNaClintake;(B)cumulativewaterintakeinduced bys.c.FURO+CAPtreatmentinratsthatreceivedbilateralinjectionsofnaloxone (nalo,100nmol/0.2␮l)orvehicle(veh,0.2␮l)combinedwithmoxonidine(moxo, 0.5nmol/0.2␮l)orvehicle(veh,0.2␮l)intotheLPBN.Resultsareexpressedas means±SE;n=numberofrats.

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Fig.5. (A)Cumulative0.3MNaClintake;(B)cumulativewaterintakeinducedbys.c. FURO+CAPtreatmentinratsthatreceivedbilateralinjectionsofbicuculline(bicu, 5.0nmol/0.2␮l)+naloxone(nalo,100nmol/0.2␮l)orvehicle(veh,0.4␮l)combined withmoxonidine(moxo,0.5nmol/0.2␮l)orvehicle(veh,0.2␮l)intotheLPBN. Resultsareexpressedasmeans±SE;n=numberofrats.

3.6. MoxonidineinjectedintoregionsoutsidetheLPBN (misplacedinjections)

Resultsfromratswithmisplacedinjections(injectionsofdrugs outside the LPBN) are shown in Table 2. Moxonidine injected outsidetheLPBNdidnotchangeFURO+CAP-induced0.3MNaCl

Table2

Waterand0.3M NaCl intakeinducedby s.c.FURO +CAP treatmentin rats thatreceived injections of bicuculline (bicu, 1.6nmol/0.2␮l),naloxone (nalo, 100nmol/0.2␮l) or vehicle (veh, 0.2␮l) combined with moxonidine (moxo, 0.5nmol/0.2␮l)orvehicle(veh,0.2␮l)outsidetheLPBN(misplacedinjections).

Treatment (ml/120min)

0.3NaClintake Waterintake

(n=3) veh+veh 3.7±2.0 14.3±1.4 veh+moxo 12.0±3.7 19.1±1.4 bicu+moxo 15.4±4.4 18.9±3.7 bicu+veh 7.7±3.3 14.2±1.2 (n=3) veh+veh 2.7±0.5 14.8±1.0 veh+moxo 5.5±2.6 16.7±1.1 nalo+moxo 4.2±1.6 11.6±1.3 nalo+veh 3.0±1.6 8.7±0.7a

a_Different_from_veh₊_veh.

Valuesaremean±S.E.M.n=numberofrats

and water intake. The only signiﬁcant effect was a reduction of FURO+CAP-induced water intakebytheinjections of nalox-one+vehicleintotheLPBN.

4. Discussion

Thepresentresultsshowthatnaloxoneorbicucullineinjected intotheLPBNpartiallyreducedtheincreasein0.3MNaClintake producedbyinjectionsof moxonidineintotheLPBN.Combined treatmentwithnaloxoneandbicucullineintotheLPBNhada simi-lareffect.NaloxoneorbicucullineinjectedaloneintotheLPBNdid notaffectFURO+CAP-induced0.3MNaClintake.Thespeciﬁcityof theLPBNasthesiteofactionfor␣2-adrenoceptoractivationwas

conﬁrmedbytheabsenceofeffectsofmoxonidinewheninjected outsidetheLPBN.Theresultssuggestthattheincreasein0.3MNaCl intakeby␣2-adrenoceptoractivationintheLPBNpartiallydepends

onGABAAandopioidreceptoractivationinthisarea.Theeffectsof

theselectiveblockadeofGABAAoropioidreceptorsweresimilar

tothoseproducedbysimultaneousblockadeofthesereceptors, whichsuggeststhatbothreceptorsarepartofthesamepathway.

BicucullineintotheLPBNatthedosesof1.6and5nmol simi-larlyreducedFURO+CAP-induced0.3MNaClintakeinratstreated withmoxonidineinthesamearea.Previousstudiesshowedthat thetreatmentwithbicuculline(1.6nmol)intotheLPBNabolished theeffectsoftheGABAAagonistmuscimol(0.5nmol)injectedinto

theLPBNon0.3MNaClandwaterintakeinﬂuidrepleterats,in FURO+CAP-treatedratsor24hsodiumdepletedrats[27,29].The injectionsofsaclofen(5nmol)intotheLPBNreduced0.3MNaCl intakeinducedbybaclofen(GABABreceptoragonist)injectedinto

theLPBNinsatiatedrats[30]andnaloxone(100nmol)injectedinto theLPBNabolishedtheingestionof0.3MNaClinducedby injec-tionsof␤-endorphinintotheLPBNinsatiatedrats[20].Therefore, thedosesoftheantagonistsusedinthepresentstudyareeffective inblockingthegabaergicoropioidreceptorsintheLPBN.

TheresultsalsoconﬁrmatleastinpartthehypothesisthatGABA and opioid receptors interact with multiple neurotransmitters independentlyfromthehydrationstatusoftheanimal[34]. Bicu-cullineandnaloxoneabolish0.3MNaClandwaterintakeinduced respectivelybymuscimoland␤-endorphininjectedintotheLPBN ofsatiated,ﬂuidreplete,rats[20,27,29].Moxonidine,however,has noeffectonsodiumorwaterintakewheninjectedintotheLPBN of satiated rats[21].It hasbeen hypothesizedthat the thresh-oldtoincreasehypertonicsodiumintakewhenGABAandopioid neurotransmissionareactivatedismuchlowerthanthatrequired toincreaseNaClintakewhen,forexample,␣2-adrenoceptorsare

activated[34].Dehydrationorthirst-relatedsignalsdecreasesuch thresholdintheLPBNallowingtheactivationof␣2-adrenoceptors

torecruitGABAandopioidmechanismsandsoincreasehypertonic NaClintake.

Recruitmentof GABAand opioidmechanismsinresponse to ␣2-adrenoceptoractivationmayalsoovercometheinhibitionof

sodiumintakeproducedbyserotonergicactivationintheLPBN.In apreviousstudy,moxonidineinjectedintotheLPBNcompletely abolishedtheinhibitionofwaterandNaClintakeproducedbythe activationoftheLPBNserotonergicmechanism[35].Thissuggests that␣2-adrenoceptorsand serotonergicreceptorsarelocatedin

thesamepostsynapticneuronsintheLPBNwitheachreceptor producingoppositeeffectsontheactivityoftheseneurons[35].

Blockadeofopioidergicand/orgabaergicreceptorsoftheLPBN inthepresentstudypartiallyreducedtheincreaseof0.3MNaCl intakecausedbytheactivationof␣2-adrenoceptorsintheLPBN,

suggesting that theactivationof ␣2-adrenoceptors mayrelease

GABAandopioidsintheLPBN.Theremainingintakeafter block-ing gabaergic and opioid mechanisms in the LPBN is probably duetothemodiﬁcationofadditionalparallelmechanismsbythe

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activationintheLPBNfacilitatesFURO+CAP-inducedwaterintake. ThereleaseofneurotransmittersthatmodulateLPBNinhibitory mechanismsarecontrolledbysignalsfromperipheralhighand lowpressurebaroreceptors,tastereceptorsorothersignalsthat reachtheLPBNthroughtheAP/NTS[5–7,16,17].FromtheLPBN, thesignalsmayreachintegrativeareasthatalsoreceive facilita-torysignalsfromforebrainareasinvolvedinthecontrolofsodium andwaterintakelike,SFO,OVLTandotherhypothalamicareas[37]. Asaconsequenceoftheactivationoftheinhibitorymechanisms, ﬂuiddepletedratsthat ingestwaterand hypertonicNaClshow reducedingestivereactionsandincreasedrejectionresponsesto anintraoralinfusionof0.3MNaCl[19].However,apreviousstudy demonstratedthatratstreatedwithmoxonidineintotheLPBN con-tinuetoshowenhancedingestivereactionsandreducedrejection responsestoanintraoralinfusionof 0.3MNaClevenafter con-suminglargevolumesof0.3MNaClandwater[19].Theseresults suggestthatmoxonidineintheLPBNpossiblyreducessometype ofinhibitorysignalsproducedasaconsequenceoftheingestionof NaClandwater.Theinhibitorysignalsaffectedbymoxonidine act-ingintheLPBNmightbethesignalsfromtastereceptorsorother visceralreceptorsthatreachtheLPBN.

Inconclusion,thepresentandpreviousstudies[19,34,35,38]

suggestthatmoxonidineactingin␣2-adrenoceptorsintheLPBN

increasestherelease ofGABAandopioidsand blocksserotonin action,reducingtheactivityoftheinhibitorymechanisms,which mightenhanceingestivereactionsandreducesrejectionresponses causedbytheingestionof0.3MNaClandwater, increasingthe intake.

Acknowledgments

TheauthorsthankSilasP.Barbosa,ReginaldoC. Queirozand SilviaFógliaforexperttechnicalassistance,SilvanaA.D.Malavolta forsecretarialassistance,AnaV.deOliveiraforanimalcareandalso PROPE–UNESP.

ThisresearchwassupportedbyBrazilianpublicfundingfrom Fundac¸ãodeAmparoaPesquisadoEstadodeSãoPaulo(FAPESP -12/01955-1)andConselhoNacionaldeDesenvolvimentoCientíﬁco eTecnológico(CNPq-478960/2013-1).

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