Chemosensory control by commissural nucleus of the solitary tract in rats

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ContentslistsavailableatSciVerseScienceDirect

Respiratory

Physiology

&

Neurobiology

jo u rn a l h om epa g e :w w w . e l s e v i e r . c o m / l o c a t e / r e s p h y s i o l

Chemosensory

control

by

commissural

nucleus

of

the

solitary

tract

in

rats

Michele

T. Favero

a,1

_{, Ana}

_C.

_Takakura

b,1

_{, Patrícia}

_M.

_de

_Paula

a

_{, Eduardo}

_Colombari

a

_,

José

V. Menani

a

_,

_Thiago

_S.

_Moreira

c,∗

a_Department_of_Physiology_and_Pathology,_School_of_Dentistry,_São_Paulo_State_University_(UNESP),_14801-903_Araraquara,_SP,_Brazil b_Department_of_{Pharmacology,}_Institute_of_Biomedical_Science,_University_of_São_Paulo_(USP),_05508-900_São_Paulo,_SP,_Brazil

c_Department_of_Physiology_and_Biophysics,_Institute_of_Biomedical_Science,_University_of_São_Paulo_(USP),_Av._Prof_Lineu_Prestes,_1524,_05508-900_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Accepted18August2011

Keywords:

Centralandperipheralchemoreflex Breathing

Sympatheticactivity NTS

Hypoxia

a

b

s

t

r

a

c

t

Thecommissuralnucleusofthesolitarytract(commNTS)isamainareathatreceivesafferentsignals involvedinthecardiovascularandrespiratorycontrollikethoserelatedtochemoreceptoractivation, however,theimportanceofthecommNTSforthecardiorespiratoryresponsestochemoreceptor activa-tionisstillcontroversial.Inthepresentstudy,weinvestigatedthecardiorespiratoryresponsestohypoxia orhypercapniainanesthetizedandconsciousratstreatedwithinjectionsoftheGABA-Aagonist musci-molintothecaudalportionofthecommNTS.MaleHoltzmanrats(280–300g)wereused.Inconscious ratsthathadastainlesssteelcannulapreviouslyimplantedintothecommNTS,theinjectionof mus-cimol(2mM)intothecommNTSreducedthepressorresponse(16±2mmHg,vs.saline:36±3mmHg) andtheincreaseinventilation(250±17ml/min/kg,vs.saline:641±28ml/min/kg)producedbyhypoxia (8–10%O2).Inurethaneanesthetizedrats,theinjectionofmuscimolintothecommNTSeliminatedthe pressorresponse(5±2mmHg,vs.saline:26±5mmHg)andtheincreaseinphrenicnervedischarge (PND)(20±6%,vs.saline:149±15%)andreducedtheincreaseinsplanchnicsympatheticnerve dis-charge(sSND)(93±15%,vs.saline:283±19%ofbaseline)producedbyhypoxia.However,muscimol injectedintothecommNTSdidnotchangehypercapnia(8–10%CO2)inducedpressorresponseorthe increaseinthesSNDorPNDinurethaneanesthetizedratsortheincreaseinventilationinconsciousrats. Thepresentresultssuggestthatthecardiorespiratoryresponsestohypoxiaarestronglydependenton thecaudalportionofthecommNTS,however,thisareaisnotinvolvedintheresponsestohypercapnia.

1. Introduction

Theneuralmechanismsinvolved in thecontrolof breathing mustberesponsivetochallengesaffectingO2,CO2,andpH lev-elsinthebody,suchasexercise,sleep,hypercapniaandhypoxia (Feldmanetal.,2003;Nattie,2006).Thephysiologicalprocessby whichbloodgasesaredetected,calledchemoreception,depends onchemicalsensorspresentintheaorticorcarotidbody (periph-eralchemoreceptors)andwithinthecentralnervoussystem(CNS) (centralchemoreceptors)(BallantyneandScheid,2001;Feldman etal.,2003;Guyenet,2008;Loeschcke,1982).

Theperipheralchemoreceptors,locatedmainlyinthecarotid body intherat,detectchanges inthepartialO2 pressure (PO₂) ortheCO2 pressure(P_CO

2)inthearterialbloodandsendsignals through theglossopharyngealnerve tothecommissuralregion ofthenucleus of thesolitarytract(commNTS)(Blessing,1997; CampanucciandNurse,2007;Colombarietal.,1996;Finleyand

∗Correspondingauthor.Tel.:+551130917764;fax:+551130917285.

E-mailaddress:tmoreira@icb.usp.br(T.S.Moreira).

1 _These_two_authors_contributed_equally_to_the_study.

Katz,1992;Sapru,1996;Smithetal.,2006).Similartothehypoxia, theintravenous(iv)injectionoflow doseofpotassium cyanide (KCN)activatestheperipheralchemoreceptorsproducing tachyp-neic,pressorandbradycardicresponsesthatareabolishedbythe bilateralligatureofthecarotidbodyarteries(Bragaetal.,2007; FranchiniandKrieger,1993;Haibaraetal.,1999;Moreiraetal., 2006).Thepressorandbradycardicresponsestoi.v.KCNarealso abolishedbyelectrolyticlesionsofthecommNTS(Colombarietal., 1996).Underanesthesia,theactivationofbreathingandtherisein sympatheticnervedischarge(SND)causedbycarotidbody stimula-tionareblockedbytheinjectionofglutamatergicantagonistsinto thecommNTS,whichsuggeststhattheprimaryafferentneurons arelikelyglutamatergic(Sapru,1996).

Detection of an increase in P_CO

2 by central and peripheral chemoreception acts to maintain stable arterial PCO₂ (Feldman etal.,2003; Smithetal.,2006).It isstill notclearwhetherthe centralchemoreceptiondependsonafewspecializedcellclusters locatedwithinthebrainstemoronmultipletypesofacid-sensitive neurons(Chernovetal., 2008;Guyenet etal.,2010; Nattieand Li, 2009). The retrotrapezoid nucleus (RTN), locus coeruleus, medullaryraphe,hypothalamicorexinergicneuronsandtheNTS neurons are the main sites suggested to beinvolved with the

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centralchemoreception(Abbottetal.,2009;Biancardietal.,2008; Deanetal.,1989;Dengetal.,2007;Johnsonetal.,2008;Moreira etal.,2007;Mulkeyetal.,2004;NattieandLi,2008;Richerson, 2004;Takakuraetal.,2006;Williamsetal.,2007).

Themainfocusofthepresentstudyistoreexaminethe ques-tionwhethertheNTS,particularlythecommissuraldivisionofthe NTScaudaltotheareapostrema,isinvolvedinchemoreception. Studiesfromtheliteraturehave suggestedthat acid-responsive neuronsare locatedin theNTSandtheacidificationof theNTS regionaltersbreathing(Deanetal.,1989;NattieandLi,2008). Addi-tionally,previousstudieshavetestedtheeffectsofthelesionsor theglutamatergicblockadeinthecommNTS,suggestingthatthis regionisessentialforthecardiorespiratoryresponsestothe periph-eralchemoreceptoractivation(Colombarietal.,1996;Sapru,1996; Bragaetal.,2007).Ontheotherhand,amorerecentstudy evalu-atedtheeffectsofmuscimolmicrodialysisinamorerostralportion ofthecommNTS,suggestingthattherostralportionofthe comm-NTSisinvolvedmainlywithrespiratoryresponsestohypercapnia (NattieandLi,2008).Basedontheassumptionsdescribedabove, inthepresentstudy,alsousingmuscimolinjectiontoblockthe neuronalactivity,weinvestigatedtheimportanceoftheneurons locatedinamorecaudalportionofthecommNTSforthe cardiores-piratoryresponseselicitedbychemoreflexactivationwithhypoxia (8–10%O2inthebreathingair)orhypercapnia(8–10%CO2inthe breathingair)inconsciousoranesthetizedrats.

2. Methods

2.1. Animals

Theexperimentswereperformedon36male Holtzmanrats weighing 300–330g. The animals were housed individually in stainless steel cages in a room with controlled temperature (24±2◦_C)_and_humidity₍₅₅_±_10%)._Lights_were_on_from_7:00_am to7:00pm.StandardGuabiratchow(Paulinia,SP,Brazil)andtap waterwereavailableadlibitum.Theexperimentalprotocolswere approvedbytheAnimalExperimentationEthicsCommitteeofthe InstituteofBiomedicalScienceofUniversityofSãoPaulo.Allefforts weremadetominimizeanimaldiscomfortandthenumberof ani-malsused.

2.2. Surgicalprocedures

2.2.1. Consciousanimals

Ratswereanesthetizedwithintraperitoneal(i.p.)injectionof ketamine(80mg/kgofbodywt)combinedwithxylazine(7mg/kg ofbodywt)andplacedinastereotaxicframe(model1760;David KopfInstruments).Astainlesssteelcannulawasimplantedintothe commNTSusingthecoordinates:15.0mmcaudaltobregma,inthe midlineand7.5mmbelowduramater.Thecannulaswerefixed tothecraniumusingdentalacrylicresinandjewelerscrews.Rats receivedaprophylacticdoseofpenicillin(30,000IU)given intra-muscularlyandasubcutaneousinjectionoftheanalgesicKetoflex (ketoprofen1%,0.03ml/rat)post-surgically.Afterthesurgery,the ratsweremaintainedinindividualboxwithfreeaccessoftapwater andfoodpellets[Guabiratchow(Paulínia,SP,Brazil)]foratleast7 daysbeforethetests.

Torecordpulsatilearterialpressure(PAP),meanarterial pres-sure(MAP)andheartrate(HR)inunanesthetizedfreelymoving rats,onedaybeforethetests,ratswereanesthetizedagainwith i.p.injectionofketamine(80mg/kgofbody wt)combinedwith xylazine(7mg/kgofbodywt)toreceiveapolyethylenetubing (PE-10connectedtoPE-50;ClayAdams,Parsippany,NJ,USA)inserted into the abdominal aorta through the femoral artery. Another polyethylenetubingwasalsoinsertedintothefemoralveinfor

drugadministration.Bothcannulasweretunneledsubcutaneously tothebackoftheratstoallowaccessinunrestrained,freelymoving rats.Wehaveevidencethattheanimalsrecoveryfromthe anes-thesiaand operativestress,because1dayafterthesurgerythe animalshadnormaldrinkandfoodintakeandnoimpairmentof motoractivity.Althoughmotoractivitywasnotquantified,visual observationintheirhomecagesandduringhandlingrevealedno apparentdifferencesinreactivity orlocomotion1 dayafterthe surgery.

2.2.2. Anesthetizedanimals

Generalanesthesia was induced with5% halothane in 100% oxygen.Theratsreceivedatracheostomyandsurgerywasdone underartificialventilationwith1.4–1.5%halothanein100% oxy-gen.Allratsweresubjectedtothefollowingpreviouslydescribed surgicalprocedures:femoralarterycannulationforarterial pres-suremeasurement,femoralveincannulationforadministrationof fluidsanddrugs,removaloftheoccipitalboneandretractingthe underlyingduramaterforinsertionofapipetteformicroinjection intothemedullaoblongataviaadorsaltranscerebellarapproach (Moreira et al., 2005,2006).All animals werebilaterally vago-tomizedtopreventanyinfluenceofartificialventilationonphrenic nervedischarge(PND).Thephrenicnervewasaccessedbya dor-solateralapproachafterretractionoftherightshoulderblade.In a groupof rats (n=7), usedto test cardiorespiratoryresponses tohypercapnia,acompletebaro-andperipheralchemoreceptor deafferentationwasperformedbysectioningthevagosympathetic trunks, thesuperior laryngealnerves and theglossopharyngeal nerves(proximaltothejunctionwiththecarotidsinusnerves). Anotherrats(n=6),usedtotestthecardiorespiratoryresponsesto hypoxia,wasagroupofbaro-andchemo-receptorintactrats,that hadthevaginervescarefullyseparatedfromthevagosympathetic trunkandselectivelytransectedbilaterally.

Splanchnicsympatheticnervedischarge(sSND)wasrecorded aspreviouslydescribed(MandelandSchreihofer,2008;Moreira etal.,2006;Takakuraetal.,2011).Therightsplanchnicnervewas isolatedviaaretroperitonealapproach,andthesegmentdistalto thesuprarenalganglionwasplacedonapairofteflon-coated sil-verwiresthathadbeenbaredatthetip(250␮mbarediameter; A-MSystems,www.a-msystems.com).Thenervesandwireswere embeddedinadhesivematerial(Kwik-CastSealant,WPI,USP),and thewoundwasclosedaroundtheexitingrecordingwires.

Upon completion of the surgicalprocedures, halothane was replacedbyurethane(1.2g/kgofbodyweight)administeredslowly i.v. All rats were ventilated with 100% oxygen throughout the experiment.Therectaltemperaturewasmaintainedat37◦_C_and theendtidal-CO2(ETCO2)weremonitoredthroughoutthe exper-imentwithacapnometer(CWE,Inc.,Ardmore,PA,USA)thatwas calibratedtwiceperexperimentagainstacalibratedCO2/N2mix. Theadequacyoftheanesthesiawasmonitoredduringa20min sta-bilizationperiodbytestingfortheabsenceofwithdrawalresponse, thelackofarterialpressurechangeandlackofchangeinthePND rateoramplitudetofirmtoepinch.Afterthesecriteriawere sat-isfied,themusclerelaxantpancuroniumwasadministeredatthe initialdoseof1mg/kgi.v.andtheadequacyofanesthesiawas there-aftergaugedsolelybythelackofincreaseinarterialpressureand PNDrateoramplitudetofirmtoepinch.Approximatelyhourly sup-plementsofone-thirdoftheinitialdoseofurethanewereneeded tosatisfythesecriteriaduringthecourseoftherecordingperiod (4h).

2.3. Intraparenchymalinjections

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7.4)waspressureinjectedintothecommNTS(50nlin5s)through single-barrelglasspipettes(20␮mtipdiameter).Injectionsinto thecommNTSweremade400␮mcaudaltothecalamus scripto-rius,inthemidlineand0.3–0.5mmbelowthedorsalsurfaceofthe brainstem.

Inconsciousfreelymovingrats,thesamedoseofmuscimolwas injectedintothecommNTSusing1␮lHamiltonsyringesconnected by polyethylenetubing (PE-10)to theinjection needle 1.5mm longerthantheguidecannulasimplantedintothebrain.

Thesolutionofmuscimolcontaineda5%dilutionoffluorescent latexmicrobeads(Lumafluor,NewCity,NY,USA)forlater histolog-icalidentificationoftheinjectionsites(Moreiraetal.,2006).

2.4. Recordingsofphysiologicalvariables

Twenty-fourhoursafterthearteryandveincannulation,when theratswerecompletelyrecoveredfromthesurgeryandadapted totheenvironmentoftherecordingroom,thearterialcatheterwas connectedtoapressuretransducer(MLT844,ADInstruments, Syd-ney,NSW,Australia)coupledtoapreamplifier(BridgeAmp,ML221, ADInstruments,Sydney,NSW,Australia) that wasconnectedto aPowerlabcomputerdataacquisitionsystem(PowerLab16/30, ML880,ADInstruments).

Therespiratoryrate(fR,breaths/min)andthetidalvolume(VT, ml/kg)inconscious,freelymovingratsweremeasuredby whole-bodyplethysmographyasdescribedindetailpreviously(Malan, 1973;Onoderaetal.,1997).Allexperimentswereperformedat roomtemperature (24–26◦_C). _In _brief,_freely_moving _rats_were keptinaplexiglassrecordingchamber(5L)thatwasflushed con-tinuouslywithamixtureof79%nitrogenand21%oxygen(unless otherwiserequiredbytheprotocol)atarateof1L/min.The concen-trationsofO2andCO2inthechamberweremonitoredon-lineusing afast-responseO2/CO2monitor(ADInstruments,NSW,Australia). Thepressuresignalwasamplified,filtered,recorded,andanalyzed off-lineusingPowerlabsoftware(Powerlab16/30,ML880/P, ADIn-struments,NSW,Australia).ThevaluesoffRandVTanalyzedwere thoserecordedfor2minbeforetheexposuretothestimulusand for2moreminattheendofeachstimulus,whenbreathing stabi-lized.ChangesinthefR,VT,andminuteventilation( ˙VE)(fR×VT;

ml/min/kg)wereaveragedandexpressedasmeans±SEM.

Themeanarterialpressure,thedischargeofthephrenicand splanchnicnervesandthetrachealO2andCO2wererecordedas previouslydescribed(Moreiraetal.,2006,2007).

Beforestartingtheexperiments,theventilationwasadjusted tohavetheETCO2at3–4%atsteady-state(60–80cycles/s;tidal volume1–1.2ml/100g).Thisconditionwasselectedbecause3–4% end-expiratoryCO2wasbelowthethresholdofthePND.Variable amountsofpureCO2wereaddedtothebreathingmixturetoadjust ETCO2tothedesiredlevel.

All analog data (ETCO2, sSND, PND and MAP) were stored ona computerviaa micro1401 digitizer(CambridgeElectronic Design)andwereprocessedoff-lineusingversion6oftheSpike 2software(CambridgeElectronicDesign)asdescribedpreviously (Takakuraet al.,2006,2011).Theintegrated phrenicnerve dis-charge(iPND)andtheintegratedsplanchnicnervedischarge(iSND) wereobtainedaftertherectificationandsmoothing(=0.015and 2s,respectively)oftheoriginalsignal,whichwasacquiredwitha 30–300Hzbandpass.

Neuralminute×volume(mvPND,ameasureofthetotalphrenic nervedischargeperunitoftime)wasdeterminedbyaveragingthe iPNDover50sandnormalizingtheresultbyassigningavalueof0to thedependentvariablerecordedatthelowlevelsofend-expiratory CO2(belowthreshold)andavalueof1atthehighestlevelofPCO₂

investigated(between9.5and10%).TheiSNDwasnormalizedfor eachanimalbyassigningthevalueof100totherestingSNAandthe valueof0totheminimumvaluerecordedeitherduringthe admin-istrationofadoseofphenylephrinethatsaturatedthebaroreflex (5␮g/kg,i.v.)oraftertheganglionicblockade(hexamethonium; 10mg/kg,i.v.).

2.5. Chemoreflexandbaroreflextests

2.5.1. Chemoreflextests

Inanesthetized rats,thehypoxiawasdoneby switchingthe breathingmixturefrom100% O2 to8–10%O2 balancedwithN2 for60s,thesameprotocolusedinapreviousstudy(Takakuraetal., 2006).ThehypercapniawasdonebyincreasingETCO2from3–3.5% to8–10%inhyperoxiacondition(100%O2)for5min(Takakura etal.,2011).

Conscious rats were maintained for at least 30min at nor-moxia/normocapnia(21%O2,79%N2,and<0.5%CO2)toadaptto thechamberenvironmentbeforestartingthemeasurementsofthe baselinearterialpressureandventilation.Hypoxiawasinducedby loweringtheO2concentrationintheinspiredairdowntoalevel of8–10%for60s.HypercapniawasproducedbyaddingCO2inthe respiratorymixtureupto8–10%CO2 for5minunderhyperoxic condition(90–92%O2),tominimizepossibleeffectsofperipheral chemoreflexactivation(Trappetal.,2008).

2.5.2. Baroreflextests

Inconsciousor anesthetizedrats,thearterialbaroreflexwas examined by raising the arterial pressure with phenylephrine (5␮g/kgofbodyweight,i.v.)andloweringthearterialpressure withsodiumnitroprusside(30␮g/kgofbody weight,i.v).These dosesofi.v.drugswerethesameusedinpreviousstudies(Moreira etal.,2005,2006;Takakuraetal.,2009).Forthei.v.injections,the drugswerepreparedinsterileisotonicsalineandthereflextests wereperformedinthesameorderwithdruginjectionsseparated bya5mininterval.

2.6. Histology

Attheendoftheexperiments,ratsweredeeplyanesthetized withhalothaneandperfusedtranscardiallywithsalinefollowedby 10%bufferedformalin(pH7.4).Thebrainwasremovedandstored inthefixativefor24hat4◦_C._The_medulla_was_cut_in₄₀_␮_m coro-nalsectionswithavibratingmicrotome(Vibratome1000SPlus– StarterCE,220V/60Hz,USA),andstoredinacryoprotectant solu-tionat−20◦_C₍_Nattie_and_Li,₂₀₀₈_)._The_injection_site_was_verified withaconventionalmultifunctionmicroscope(OlympusBX50F4, Japan).Thesectionalignmentbetweenthebrainswasdonerelative toareferencesection.ToalignthesectionsaroundNTSlevel,the mid-areapostremalevelwasidentifiedineachbrainandassigned thelevel13.8mm(Bregma−13.8mm)accordingtotheatlasof PaxinosandWatson(1998).Thecoordinatesofsectionsrostraland caudalofthisreferencesectionwerecalculatedwithrespecttothe referencesection,usingthenumberofinterveningsectionsandthe sectionthickness.

2.7. Statistics

ThestatisticalanalysiswasdonewithSigmaStatversion3.0 (JandelCorporation,PointRichmond,CA).Thedataarereportedas means±standarderrorofthemean(SEM).Thet-testoroneway parametricANOVAfollowedbytheNewman–Keulsmultiple com-parisonstestwereusedasappropriate.Thesignificancewassetat

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Fig.1.(A)PhotomicrographshowingthetypicalinjectionsiteintothecommNTS inconsciousrats(arrows).Scale=1mm.(B)Thecenterofmuscimolinjectionsinto thecommNTSinthedifferentratstestedrepresentedonasinglesection(Bregma

−14.3mm,accordingtoPaxinosandWatson,1998).Scale=1mm.cc,centralcanal; IO,inferiorolive;py,pyramide;Sp5,spinaltrigeminaltract;XII,hypoglossalnucleus.

3. Results

3.1. Histologicalanalysis

Muscimolinjections into thecommNTS were located about 400␮mcaudaltothecalamusscriptoriusasillustratedinFig.1Aand B.Asingleinjectionofmuscimolwasadministeredinornearthe midlineasrepresentedinFig.1B.Basedontheareaofthe distribu-tionofthefluorescentmicrobeads,theinjectatespreadbilaterally (approximately500␮mfromtheinjectioncenter)andalittlelessin therostrocaudaldirection(approximately300␮mfromthe injec-tioncenter).

3.2. Cardiorespiratoryresponsestohypoxiainanesthetizedor consciousratstreatedwithmuscimolintothecommNTS

Inurethane-anesthetizedrats,incontrolconditions(aftersaline injectedintothecommNTS),abriefperiodofhypoxia(8–10%O2 inthebreathingairfor60s)producedaninitialincreaseinMAP (26±5mmHg)inthefirst5–10sfollowedbyadecreaseinMAP (−47±6mmHg)thatreachthemaximumattheendoftheperiod ofhypoxia (Fig. 2A1 and B1). In theseconditions, hypoxiaalso increasedsSND(283±19%ofthebaseline)andmvPND(calculated astheproductofphrenicnervefrequencyandamplitude–f×a

–ameasureofthetotalphrenicneuraloutput)(149±25%ofthe baseline)(Fig.2A1,CandD).

Injectionofmuscimol(100pmol/50nl)intothecommNTSdid notchangerestingMAP(112±3mmHg,vs.saline:110±5mmHg,

p>0.05),sSNDandmvPND(Fig.2A2).ThePNDamplitude(98±6% ofcontrol;p>0.05)andduration(from0.48±0.02to0.47±0.05s,

p>0.05)alsodidnotchange.Muscimolinjectedwithinthe comm-NTS blocked the pressor response (5±2mmHg, p<0.01) and reducedsympathoexcitation(93±15%ofthebaseline,p<0.01)and theincreaseinPND (20±6%ofthebaseline, p<0.01) produced byhypoxia (Fig.2A2,2B–D). Muscimolinto thecommNTSalso

increasedthehypotensionproducedby60sofhypoxiain anes-thetizedrats(−63±4mmHg,p<0.05)(Fig.2A2andB).

In consciousrats,in controlconditions (after salineinjected into thecommNTS), 60s ofhypoxia (8–10% O2 in theinspired air)undernormocapniaincreasedMAP(36±3mmHg),fR(60±4 breaths/min),VT(4±0.3ml/kg)and ˙VE (641±28ml/min/kg)and

reduced HR (−96±6bpm) (Fig. 3A–E). Injection of muscimol (100pmol/50nl) into the commNTS, in conscious rats, did not change resting MAP (113±6mmHg, vs. saline: 117±5mmHg,

p>0.05)andHR(335±21bpm,vs.saline:341±18bpm,p>0.05). MuscimolinjectionwithinthecommNTSreducedtheincreaseon MAP(16±2mmHg,p<0.05),fR(26±3breaths/min,p<0.05),VT (1.8±0.2ml/kg,p<0.05)and ˙VE(250±17ml/kg/min,p<0.01)and

blockedthebradycardia(1±2bpm,p<0.01)producedbyhypoxia (Fig.3A–F).

3.3. Cardiorespiratoryresponsestohypercapniainanesthetized orconsciousratstreatedwithmuscimolinjectedintothe commNTS

Inurethane-anesthetizedrats,incontrolconditions(aftersaline injectedintothecommNTS),hypercapnia(from5%to10%CO2for 5min)producedanimmediatehypotension(−22±4mmHg)that wasgraduallyreducedwithMAPreturningtoorslightlyabove con-trollevelsattheendofhypercapnia.Immediatelyafterstopping hypercapnia(returningto5%CO2),MAPincreased(27±5mmHg) andreturnedtocontrolvaluesafteraround5min(Fig.4A1andB). Incontrolcondition,hypercapniaalsoincreasedsSND(108±13% ofbaselineat5%CO2)andmvPND(111±8%ofthebaselineat5% CO2)(Fig.4A1,CandD).Injectionofmuscimol(100pmol/50nl) intothecommNTSdidnotaffectrestingMAP,sSNDormvPNDor hypercapnia-inducedincreaseinMAP,sSNDormvPNDinurethane anesthetizedrats(Fig.4A2,B–D).

Inconsciousrats,incontrolconditions(aftersalineinjectedinto thecommNTS),hypercapnia(8–10%CO2 intheinspiredair)for 5minunderhyperoxiccondition(92–98%O2,tominimizepossible effectsofperipheralchemoreflexactivation)increasedfR(55±6 breaths/min), VT (3.7±0.4ml/kg) and ˙V_E (611±19ml/min/kg), however,producednosignificantchangeinMAP(5±2mmHg)or HR(−4±3bpm)(Table1).Injectionofmuscimol(100pmol/50nl) intothecommNTSproducednochangeinrestingMAP,HRandVE oroncardiorespiratoryresponsestohypercapniainconsciousrats (Table1).

3.4. Cardiovascularresponsestobaroreflexactivationinrats treatedwithmuscimolinjectedintothecommNTS

Injectionsofmuscimol(100pmol/50nl)withinthecommNTS inanesthetizedratsdidnotaffectthepressorresponseand sympa-thoinhibitiontoi.v.phenylephrine(PHE,5␮g/kgofbodyweight)or thehypotensionandsympathoactivationtoi.vinjectionofsodium nitroprusside(SNP,30␮g/kgofbodyweight)(Table2).PHEorSNP i.v.didnotmodifymvPND(Table2).

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Fig.2.(A)Typicalrecordingsofarterialpressure(AP),splanchnicsympatheticnervedischarge(sSND)andphrenicnervedischarge(PND)inoneratrepresentativeofthe groupsubmittedto60sofhypoxia(8–10%O2)afterinjectionof(A1)salineor(A2)muscimol(100pmol/50nl)intothecommNTSunderanesthesia.(B,CandD)Changesin

meanarterialpressure(MAP),sSND(sSND)andmvPND(mvPND),respectively,producedby60sofhypoxiaafterinjectionofsalineormuscimolintothecommNTSin anesthetizedrats.*Differentfromsaline(p<0.05);n=6rats/group.

Fig.3. (A)Typicalrecordingsofbreathing,arterialpressure(AP),meanarterialpressure(MAP)andheartrate(HR)inoneratrepresentativeofthegroupsubmittedto60s ofhypoxia(8–10%O2)afterinjectionofsalineormuscimol(100pmol/50nl)intothecommNTSinconsciousrats.(B–F)Changesinmeanarterialpressure(MAP),heartrate

(HR),ventilation( ˙VE),respiratoryfrequency(fR)andtidalvolume(VT),respectively,producedbyhypoxia(8–10%O2)before(salineinjection)andaftermuscimolinjection

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Fig.4. (A)Typicalrecordingsofarterialpressure(AP),splanchnicsympatheticnervedischarge(sSND)andphrenicnervedischarge(PND)inoneratrepresentativeofthe groupsubmittedto5minofhypercapnia(steppingETCO2from5to10%)after(A1)salineor(A2)injectionofmuscimol(100pmol/50nl)intothecommNTSunderanesthesia.

(B–D)Changesinmeanarterialpressure(MAP),sSND(sSND)andmvPND(mvPND),respectively,producedbyhypercapniaafterinjectionofsalineormuscimolinto thecommNTSinanesthetizedrats.*Differentfromsaline(p<0.05);n=6rats/group.

Table1

CardiorespiratoryresponsestohypercapniainconsciousratstreatedwithmuscimolinjectedintothecommNTS.

Treatment n MAP(mmHg) HR(bpm) fR(breaths/min) VT(ml/kg) V˙E(ml/min/kg)

Saline(control) 6 +5±2 −4±3 +55±6 +3.7±0.4 +611±19

Muscimol 6 +5±3 −6±2 +53±5 +3.8±0.5 +668±28

Valuesaremeans±SEM.n=numberofrats.MAP,changesinmeanarterialpressure;HR,changesinheartrate;fR,changesinrespiratoryfrequency;VT,changesin tidalvolume;V˙E,changesinventilation.Muscimol(100pmol/50nl).Hypercapnia(8–10%CO2intheinspiredair)for5minunderhyperoxiccondition(100%O2).

Table2

CardiovascularresponsestophenylephrineandsodiumnitroprussideinanesthetizedratstreatedwithmuscimolinjectedintothecommNTS.

Treatment n MAP(mmHg) sSND(%) mvPND(%)

PHE SNP PHE SNP PHE SNP

Saline(control) 5 +32±3 −34±5 −98±3 +101±6 +9±9 +6±13

Muscimol 6 +34±7 −38±4 −94±8 +97±13 +10±4 +8±11

Valuesaremeans±SEM.n=numberofrats.PHE,phenylephrine(5␮g/kgofbodyweight);SNP,sodiumnitroprusside(30␮g/kgofbodyweight);MAP,changesinmean arterialpressure;sSND,changesinsplanchnicsympatheticnervedischarge;mvPND,changesinphrenicnervedischarge.

PHEandSNPi.v.,respectively,didnotchange ˙V_Einconsciousrats (Table3).

3.5. SpecificityofcommNTSasthesitefortheeffectsofmuscimol

Injections of muscimol outside the commNTS (n=4) did not change the pressor (25±4mmHg, p>0.05), sympathetic (270±15%ofbaseline,p>0.05)andphrenic(136±9%ofbaseline,

p>0.05) responsesevoked byperipheralchemoreflexactivation withbrief period of hypoxia in anesthetized rats. In conscious rats,theinjectionofmuscimoloutsidecommNTS(n=7)produced nochangeonpressor(33±6mmHg),fR(54±9breaths/min),VT (4.2±0.4ml/kg)and ˙VE(631±33ml/min/kg)responsesandonthe

bradycardia(−84±11bpm)producedbyhypoxia.

4. Discussion

Thepresentresultsprovidefunctionalevidencethatthecaudal portionofthecommNTSisessentialforthepressorresponseand theincreaseintheSNDandbreathingproducedbyhypoxiain con-sciousoranesthetizedrats.However,theresultsshownoevidence thatthisportionoftheNTSisinvolvedinmediating cardiorespi-ratoryresponsestohypercapnia.Inaddition,theinhibitionofthe caudalcommNTSneuronsdidnotmodifytheresponsesproduced bybaroreflexactivationaspreviouslydemonstrated(Moreiraetal., 2009).

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Table3

CardiovascularresponsestophenylephrineandsodiumnitroprussideinconsciousratstreatedwithmuscimolinjectedintothecommNTS.

Treatment n MAP(mmHg) HR(bpm) V˙E(ml/kg/min)

PHE SNP PHE SNP PHE SNP

Saline(control) 6 +49±5 −28±6 −59±8 +88±4 +21±8 +11±5

Muscimol 6 +54±6 −31±5 −44±11 +93±8 +25±7 +18±8

Valuesaremeans±SEM.n=numberofrats.PHE,phenylephrine(5␮g/kgofbodyweight);SNP,sodiumnitroprusside(30␮g/kgofbodyweight);MAP,changesinmean arterialpressure;HR,changesinheartrate;V˙E,changesinventilation.

centrallymediatedvasoconstrictionthatdependson chemorecep-torand sympatheticactivation.Previousstudieshavesuggested thatanestheticsmayaffectneurotransmissiononthebrainstem andconsequentlyreflexresponses(Accorsi-Mendonc¸aetal.,2007; MachadoandBonagamba,1992).Therefore,inthepresentstudy, cardiorespiratory responses to hypoxia and hypercapnia were testedinanesthetizedratsandalsoinconsciousratsusing whole-bodyplethysmography,whichallowssimultaneousmeasurements ofrespiratoryandcardiovascularresponseswithoutrestrainingthe animal(Biancardietal.,2008;Bracciallietal.,2008;dePaulaand Branco,2005).

In urethane-anesthetized,vagotomized andartificially venti-latedrats,incontrolconditions,hypoxiaorhypercapniaproduced a dual response onarterialpressure. The hypoxiaproduced an initialincreaseinMAPinthefirst5–10sthatwasfollowedbya decreaseinMAPthatreachtheminimumvalueattheendofthe periodofhypoxia.Thehypercapniareducedarterialpressurein thefirstminutefollowedbyanincreaseattheendofthe5-min hypercapnia.ThehypoxiaorhypercapniarapidlyincreasedPND andgraduallyincreasedsSNDwhichreachesthemaximumatthe endofthetest.Inconsciousrats,incontrolconditions,hypoxia orhypercapniaincreasedventilationandhypoxiaincreasedMAP, whereashypercapniaproducednochangeinMAP.

The blockade of neuronal activity with muscimol injection intothecommNTSalmostabolishedthepressor,sympatheticand phrenicresponsestohypoxia inanesthetized ratsand partially reducedthepressorandrespiratoryresponsestohypoxiain con-sciousrats,whereasthesametreatmentinthecommNTSproduced nochangesinthecardiorespiratoryresponsestohypercapniain consciousoranesthetizedrats.Therefore,inanesthetizedor con-scious rats, it seems that chemoreflex-mediated cardiovascular andrespiratoryresponsestohypoxiaarestronglydependenton caudalcommNTSmechanisms.However,inconsciousrats, neu-ronal blockade in the commNTS with muscimol only partially reducedcardiorespiratoryresponsestohypoxia.Theeffectsof mus-cimolinjectedintothecommNTSinconsciousratsaresimilarto those previously demonstrated in theworking heart-brainstem preparationaftercombiningglutamatergicandpurinergic recep-torblockadeinthecommNTS(Bragaetal.,2007),whichsuggest thatinthiscasecardiorespiratoryresponsestohypoxiaarealso mediatedbysignalsthatarisefromothercentralsitesnotrelated tocommNTS.

Apreviousstudyshowedthatelectrolyticlesionsofthe comm-NTSabolishedthepressorandbradycardicresponsestoperipheral chemoreceptor activationwith i.v. injectionof KCN(Colombari etal.,1996).Itisinterestingtonotethattheresultsofthepresent studyshowedthatmuscimolintothecommNTSonlyreducedthe pressor responsesto hypoxiain consciousrats, whereas inthe previousstudyelectrolyticlesionsofthecommNTSabolishedthe pressorresponsetoi.v.KCN.Thesedifferencesalsosuggestthat, inconsciousrats,besidestheactivationofperipheral chemore-ceptors,additionalmechanismsareactivatedbyhypoxia,probably centrally,thatdonotdependoncommNTS(Colombarietal.,1996). Although the cardiorespiratory responses to hypoxia are stronglydependentoncommNTSneuronactivation,thepresent

resultssuggestthatthesamecommNTSneuronsinvolvedinthe responses tohypoxia are not involved in thecardiorespiratory responsestohypercapnia.MuscimolinjectionsintothecommNTS didnotchangetheincreaseinarterialpressure,SNDor breath-ingproducedbyhypercapnia.However,apreviousstudyshowed thatitispossibletoreducetherespiratoryresponsesto hypercap-niabymuscimolmicrodialysisinthecommNTS,suggestingthat commNTSmaydetectCO2 (NattieandLi,2008).Thesamestudy alsoshowedthatmuscimolmicrodialysisinthecommNTSdidnot affectrespiratoryresponsestohypoxiawhenratsweretestedat roomtemperatureof24◦_C,_the_same_room_temperature_that_rats were exposed in thepresent study. Therefore, thepresent and thepreviousstudyshowdifferenteffectsofthecommNTS inhi-bitionwithmuscimolinthecontroloftherespiratoryresponsesto hypoxiaorhypercapnia.Possiblereasonsforthedifferentresults arethedifferencesinthesiteofmicrodialysis/injectionsintothe commNTS,thevolumeofmicrodialyis/injectionsandthe concen-trationofmuscimolreleasedinthecommNTS.Inthepreviousstudy (NattieandLi,2008),microdialysisprobesreleasedmuscimolinto thecommNTSbilaterallyattheleveloftheareapostrema,whereas inthepresentstudyjustoneinjectionwasperformedinthe mid-linearound400␮mcaudaltotheareapostrema,i.e.,theprevious studytestedtheeffectsofmuscimolinamorerostralportionof thecommNTSandthepresentstudyinamorecaudalportionof thecommNTS.Although,differentsitesofinjections/microdialysis seemtobethemainreasonforthedifferentresults,inthe pre-viousstudy,theconcentrationofmuscimolwas0.5mMandthe volumeofmicrodialyiswas4␮l/mincontinuouslythroughoutthe entireexperiment (Nattieand Li,2008), whereasinthepresent studytheconcentrationofmuscimolwas2mMandavolumeof 50nlwasinjectedinasingleinjection.Althoughinbothstudies thenomenclatureisthesame(commissuralNTS),theydidnottest thesamearea/neurons:thepresentstudytestedamorecaudal por-tionofthecommNTSandthepreviousstudy(NattieandLi,2008) testedamorerostralpartofthecommNTS.Therefore,basedon thepresentandthepreviousstudy(NattieandLi,2008)itis possi-bletosuggestthatdifferentpartsofthecommNTSareinvolvedin therespiratoryresponsestohypoxiaandhypercapnia.According tothepresentresults,amorecaudalportionofthecommNTSis involvedincardiorespiratoryresponsestohypoxia,whereasa pre-viousstudysuggeststhatamorerostralportionofthecommNTS isthesiteofthepH-sensitivecells oftheNTSimportantmainly for therespiratory responsestohypercapnia.Thesesuggestions arecoherentwiththemassiveprojectionsfromthecommNTSto therespiratorycentralpatterngenerator(CPG)(Aicheretal.,1996; EzureandTanaka,2004;KoshiyaandGuyenet,1996;Kubinetal., 2006)thatprobably conveyimportantsignals fromcentraland peripheralchemoreceptors.

Acknowledgements

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