Neural mechanisms underlying respiratory rhythm generation in the lamprey

(1)

RespiratoryPhysiology&Neurobiology224(2016)17–26

ContentslistsavailableatScienceDirect

Respiratory

Physiology

&

Neurobiology

j o ur na l ho me p ag 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

Neural

mechanisms

underlying

respiratory

rhythm

generation

in

the

lamprey

Fulvia

Bongianni

∗

,

Donatella

Mutolo,

Elenia

Cinelli,

Tito

Pantaleo

DipartimentodiMedicinaSperimentaleeClinica,SezioneScienzeFisiologiche,UniversitàdegliStudidiFirenze,VialeG.B.Morgagni63,50134Firenze,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Accepted5September2014 Availableonline16September2014 Keywords:

Respiratoryrhythmgeneration Controlofbreathing Evolutionofbreathing GABAAandglycinereceptors

Glutamatergictransmission

a

b

s

t

r

a

c

t

Theisolatedbrainstemoftheadultlampreyspontaneouslygeneratesrespiratoryactivity.The para-trigeminalrespiratory group (pTRG),theproposed respiratorycentral patterngenerator,hasbeen anatomicallyandfunctionallycharacterized.Itissensitivetoopioids,neurokininsandacetylcholine. Excitatoryaminoacids,butnotGABAandglycine,playacrucialroleintherespiratory rhythmogene-sis.Theseresultsarecorroboratedbyimmunohistochemicaldata.WhileonlyGABAexertsanimportant modulatorycontrolonthepTRG,bothGABAandglycinemarkedlyinﬂuencetherespiratoryfrequencyvia neuronsprojectingfromthevagalmotoneuronregiontothepTRG.Noticeably,theremovalof GABAer-gictransmissionwithinthepTRGcausestheresumptionofrhythmicactivityduringapneainducedby blockadeofglutamatergictransmission.ThesameresultisobtainedbymicroinjectionsofsubstanceP ornicotineintothepTRGduringapnea.Theresultspromptedustopresentsomeconsiderationsonthe phylogenesisofrespiratorypatterngeneration.Theymayalsoencouragecomparativestudiesonthe basicmechanismsunderlyingrespiratoryrhythmogenesisofvertebrates.

1. Introduction

Thevertebratenervoussystemisorganizedinasimilarway throughoutvertebrate phylogenesis,althoughthelevel of com-plexity increases. Lampreys are jawless vertebrates known as cyclostomes.Theyhavechangedcomparativelylittleduring evo-lution,andbecameseparatedfromthemainvertebrateline560 millionyearsago(KumarandHedges,1998).Thelampreycentral nervoussystemcanberegardedasavertebrateprototype,with theexperimentaladvantagethatithasfewerneuronsthanhigher vertebratesandcanbemaintainedinvitro.Studiesonneuronal networksof thelampreyprovedtobehighly usefultoprovide insightsintothebasicmechanismsofcentralpatterngenerators (CPGs)ofrhythmicactivities,suchaslocomotionandrespiration (Grillner,2003,2006).Thelampreymodelhasbeenusedformany yearstoidentifythecellularmechanismsinvolvedinthegeneration andcontroloflocomotioninextremedetailand,morerecently,to investigatetheneuralmechanismsunderlyingrespiratoryrhythm generation.Thebasicfeaturesoftheneuralorganizationaswell asthoseofrhythmogenicnetworkshavebeenconserved through-outvertebrateevolution(Grillner,2003;Mutoloetal.,2007,2010;

∗ Correspondingauthor.Tel.:+390552751608;fax:+390554379506. E-mailaddress:fulvia.bongianni@uniﬁ.it(F.Bongianni).

Robertsonetal.,2007;Kinkead,2009;Ericssonetal.,2011,2013; Stephenson-Jonesetal.,2011,2012a,2012b;Cinellietal.,2013).

This review examines the main characteristics of the lam-preyrespiratorynetworkanddescribesrecentresultsconcerning respiratory rhythm generation and the relevant role of some neurotransmitters and neuromodulators. We also consider the characteristicsofrespiratoryCPGsduringvertebrateevolutionand possibleevolutionarytrendsinrespiratoryrhythmgeneration.

2. Generalfeaturesofthelampreyrespiratorysystem

In theadultlamprey, breathing isproduced bysynchronous contractionsofthebranchialmusclesthatforcewateroutofthe gillopenings;theinhalationphaseispassiveandisproducedby theelasticrecoilofcartilaginousbasketssurroundingthegillsacs (Rovainen,1977,1979).Theisolatedbrainstemoftheadultlamprey spontaneouslygeneratesrespiratoryneuronalactivityinvitro;this activitycloselyresemblesthatunderlyingtherespiratory behav-iorofintactanimalsandpersistsaftertransectionsofthebrainat boththeobexandisthmuslevel(Rovainen,1977,1983;Thompson, 1985;Russell,1986).Thus,boththeneuralnetworkresponsiblefor respiratoryrhythmgenerationandrespiratorymotoneuronsare locatedwithinthebrainstem.Theresultsobtainedinthis prepa-rationcontributetoimprovecurrentknowledgeonthesynaptic transmissionwithintherespiratorynetworkofthelampreyand, http://dx.doi.org/10.1016/j.resp.2014.09.003

(2)

18 F.Bongiannietal./RespiratoryPhysiology&Neurobiology224(2016)17–26

Fig.1.LocalizationofthepTRG.(A)Schematicillustrationofadorsalviewofthelampreymesencephalon/rhombencephalonshowingthelevelsofthecoronalsections illustratedinBandC(arrows)andthelocationofthepTRG(pinkarea).(B)Photomicrographofatransversesectionoftherostralrhombencephalonshowingthelocation ofanhorseradishperoxidaseinjectionintothepTRG.(C)Photomicrographofatransversesectionsoftherhombencephalonshowingthelocationofﬂuorescentbeads microinjectedintothepTRG(green).ARRN,anteriorrhombencephalicreticularnucleus;I1,isthmicMüllercell;nVm,motorrootofthetrigeminalnerve;nVs,sensoryrootof

thetrigeminalnerve;pTRG,paratrigeminalrespiratorygroup;SL,sulcuslimitansofHis.V,trigeminalmotornucleus;VII,facialmotornucleus;IX,glossopharyngealmotor nucleus;X,vagalmotornucleus.BandCadaptedfromMutoloetal.(2007)andCinellietal.(2014),respectively.

possibly, to obtainnew hints for furtherinvestigations onthe basicneuralmechanismsoperatingintherespiratorynetworkof highervertebrates,includingmammals.Thevastmajorityof respi-ratory motoneurons are located in the facial, glossopharyngeal and,especially,vagalnuclei,whiletheneuralaggregate respon-sibleforrespiratoryrhythmgenerationappearstobelocatedin aregionrostrolateraltothetrigeminalmotornucleus(Rovainen, 1977,1979,1983,1985;Thompson,1985;Russell,1986;Bongianni etal.,1999,2002,2006;Guimondetal.,2003;Marteletal.,2007; Mutoloetal.,2007).Mutoloetal.(2007)reportedthatopioidshave amodulatoryroleintherespiratorynetworkand,inparticular,that microinjectionsofthe␮-opioidreceptoragonistDAMGOatsites rostrolateraltothetrigeminalmotornucleusabolishthe respira-toryrhythm.Theseapneicresponsessupportthehypothesisthat thisspecificopioid-sensitiveregionlikelyhasapivotalrolein respi-ratoryrhythmogenesis.Mutoloetal.(2007)proposedtonamethis areatheparatrigeminalrespiratorygroup(pTRG).Theresultson thedepressanteffectsofopioidsonthelampreyrespiratory activ-ityalsoimply that theinhibitoryrole of opioidsonrespiration ispresentatanearlystageofvertebrateevolution. Respiration-relatedneuronswithdifferentfiringpatternsarepresentinthe pTRG(Mutoloetal.,2007,2010),thuscorroboratingourhypothesis ontheinvolvement ofthepTRG inrespiratory rhythm genera-tion.The differentdischarge patternsencountered in thepTRG may suggest different neuronal functions, but at present any attempttoascribeaspecificroletoeachtypeofneuronsisonly speculative.

3. AnatomicalandfunctionalcharacterizationofthepTRG An anatomical and functional characterization of the pTRG region has been recently provided (Cinelli et al., 2013, 2014).

By retrograde labeling, we found neurons located in the isth-micperiventricularcelllayerwithaxonalprojectionstothevagal motoneuronregion.Projectingneuronscanbeeasilyidentiﬁedby anatomicallandmarks,i.e.theyarelocatedinadorsalaspectofthe anteriorrhombencephalicreticularnucleus,atthelevelofthe isth-micMüllercellI1,closetothesulcuslimitansofHis.Thisregion

correspondscloselytothepTRGasdeﬁnedinourprevious stud-ies(Mutoloetal.,2007,2010,2011).NeuronslocatedinthepTRG projecttothe ipsilateraland contralateral vagal motor nucleus aswellastothecontralateralpTRG(Rovainen,1985;Thompson, 1985; Russell, 1986; Gariépy et al., 2012; Cinelli et al., 2013). Theresultsobtainedwithmicroinjectionsofseveralneuroactive drugs,suchasDAMGO,substanceP(SP),acetylcholine(ACh), glu-tamateor GABAreceptor agonistsand antagonists,exactlyinto thisregion(Mutoloetal.,2007,2010,2011;Cinelli etal.,2013, 2014)helptoidentifyandcharacterizethepTRGandtosupport thenotionthatitcorrespondstotherespiratoryCPG(seebelow). Aschematicrepresentationofadorsalviewofthelamprey mesen-cephalon/rhombencephalonshowingtherespiration-relatedareas alongwithphotomicrographsoftransversesectionsillustratingthe localizationofthepTRGisreportedinFig.1.

4. Glutamatergicmechanismsintherespiratory rhythmogenesis

Endogenouslyreleasedexcitatoryaminoacidsplayacrucialrole inthelampreyrespiratory rhythmogenesisactingonionotropic receptors(Bongiannietal.,1999)andexertamodulatoryroleon respiratoryactivityviametabotropicreceptors(Bongiannietal., 2002).Thesuppressionofrespiratoryactivitycausedbybath appli-cation of ionotropic glutamate receptor antagonists (Bongianni etal.,1999;Mutoloetal.,2011)ismimickedbymicroinjections

(3)

F.Bongiannietal./RespiratoryPhysiology&Neurobiology224(2016)17–26 19

Fig.2.ExamplesofrespiratoryresponsesevokedbyblockadeofionotropicglutamatereceptorsandbyblockadeoractivationofthepTRGregion.(A)Suppressionof respiratoryrhythmicactivity∼20minafterbathapplicationofamixtureof20␮MCNQXand100␮MD-AP5.Aschematicillustrationofadorsalviewofthelamprey mesencephalon/rhombencephalonintheperfusedrecordingchamberisshown.(B)Suppressionofrespiratoryrhythmicactivity∼1minafteraunilateralmicroinjectionof amixtureof1mMCNQXand5mMD-AP5intothepTRGregion.(C)Increasesinrespiratoryfrequencyandpeakvagalactivity∼2minafteraunilateralmicroinjectionofa mixtureof1mMAMPAand2mMNMDAintothepTRG.Thesitewheredrugsweremicroinjected(pinkarea)isshownonaschematicillustrationofadorsalviewofthe lampreymesencephalon/rhombencephalon.VA,rawvagalnerveactivity;IVA,integratedvagalnerveactivity.ModiﬁedfromCinellietal.(2013).

ofthese drugsintothepTRG (Martel etal.,2007; Cinelli etal., 2013,2014), thus supportingthehypothesis that this regionis crucialforrespiratoryrhythmgeneration.Examplesoftheapnea causedbyionotropicglutamatereceptorblockadearereportedin Fig.2AandB.Inagreementwiththisﬁndingandwiththeviewthat changesinrespiratoryfrequencyareduetoanactiononthecentral mechanismsgeneratingtherespiratoryrhythm(Grayetal.,1999; Feldmanand DelNegro, 2006;Bongiannietal.,2008), wehave shownthatmicroinjectionsofglutamateagonistsintothepTRG causeamarkedincreaseinrespiratoryfrequencyassociatedwith increasesinpeakamplitudeanddurationofvagalbursts(Cinelli etal.,2013).Fig.2Cshowsincreasesinrespiratoryfrequency fol-lowinga microinjectionofglutamate receptoragonistsintothe pTRG.

Recently, evidence has been provided that pTRG neurons, retrogradely labeled from the vagal motoneuron region, are immunoreactiveforglutamate,thusshowingthatglutamatergic transmissionmediatestheexcitatoryinputtovagalmotoneurons (Fig.3).InthepTRGregionthere arealsoglutamate-expressing cells, not retrogradely labeled, that may represent interneu-rons of the respiratory CPG (Cinelli et al., 2013). Accordingly, a recent studyreported thepresence of numerous glutamater-gic neurons in the isthmic region of the lamprey where we haveidentiﬁedretrogradelylabeledneurons(Villar-Cervi ˜noetal., 2012).

5. GABAergicandglycinergicmechanismsinthe respiratorynetwork

Both GABA-and glycine-mediated inhibition are not essen-tialforrespiratoryrhythmgeneration(Rovainen,1983;Bongianni etal.,2006),butmayrepresentmechanismssuitabletoregulate

the excitabilitylevel of the respiratory network (Mutoloet al., 2011;Cinellietal.,2014).BlockadeofGABAAand/orglycine

recep-tors by bath application of the appropriate antagonists causes potent excitatory effects on respiration by increasing the fre-quencyandamplitudeofvagalbursts.Ontheotherhand,GABAB

receptorantagonistsappliedtothebathinduceslightdecreases in the respiratory frequency (Bongianni et al., 2006). Antago-nist microinjections into thepTRG revealed that an important modulatory control is exerted atthat level byGABA acting on GABAA and GABABreceptors, whilea glycinergicmodulationis

lacking(Cinellietal.,2014).GABABantagonismwithinthepTRG

induces only modest decreases in respiratory frequency prob-ably due toa presynaptic mechanism. The blockade of GABAA

receptorsincreasesrespiratorynetworkexcitabilityapparentlyby acting onthemechanismsgenerating respiratory bursts within thepTRGwithlittleinﬂuenceontheinterburstperiod.These out-comessuggestthatGABAreceptorsmayconceivablycontribute toaninhibitorycontroloftheexcitabilityof pTRGneuronsand togeneratea moreregular respiratoryrhythm.Consistent with our interpretation, an increase in neuronal excitability of the preBötzingercomplex(preBötC),theproposedmammalian respi-ratory CPG, has been observed following a blockade of GABAA

receptors both in invitro and in vivopreparations (Kamet al., 2013).Furthermore,duringtheapneacausedbybathapplicationof ionotropicglutamatereceptorantagonists,theblockadeofGABAA,

but not glycine receptorswithin thepTRG causesthe resump-tion of rhythmicactivity(Cinelli et al., 2014), thusunderlining theprominentrole ofGABAergicmechanismswithinthepTRG. TheseeffectsareillustratedinFig.4AandB.Theinhibitory con-trolofpTRGneuronsisalsoemphasizedbytheprolongedapnea caused by the GABAA agonistmuscimol microinjected into the

(4)

Fig.3. DistributionofglutamateimmunoreactivityinthepTRG.(A)Schematicillustrationofadorsalviewofthelampreymesencephalon/rhombencephalonshowingthe siteofaNeurobiotininjectionintothevagalnucleus(green)andthelocationofretrogradelylabeledneuronswithinthepTRG(greencircles).(B)Photomicrographofa transversesectionattheleveloftherostralrhombencephalonshowingretrogradelylabeledneurons(merged,Neurobiotingreen+glutamateimmunoreactivityredsignals) inthepTRGregionafteraNeurobiotininjectionintothevagalmotoneuronpool.(C)Photomicrographsatahighermagniﬁcationoftheportionofthetransversesection indicatedbythewhiterectangleinBshowingretrogradelylabeledneurons,glutamateimmunoreactivityandmergedimageatthelevelofthepTRGregion.Retrogradely labeledneuronsdisplayingimmunoreactivityforglutamateareindicatedbywhitearrows.ARRN,anteriorrhombencephalicreticularnucleus;I1,isthmicMüllercell;pTRG,

paratrigeminalrespiratorygroup;V,trigeminalmotornucleus;VII,facialmotornucleus;IX,glossopharyngealmotornucleus;X,vagalmotornucleus.Scalebars:B,200␮m; C,100␮m.ModiﬁedfromCinellietal.(2013).

lackingGABA,butaresurroundedbyGABA-immunoreactive struc-tures(Fig.4D).

Interestingly,increasesinrespiratoryfrequencycausedbybath applicationofbicucullineorstrychnine(Rovainen,1983;Bongianni etal.,2006)aremimickedbymicroinjectionsofthesedrugsinto the vagal motoneuron region (Cinelli et al., 2014). The effects causedby blockade ofGABAA and glycine receptorswithinthe

vagalmotoneuronregionarereportedinFig.5.Evidencehasbeen providedthatneuronswithinthisregionreceiveGABAergicand glycinergicinputsandareinvolvedintheregulationofrespiratory frequencyviaascendingexcitatoryprojectionstothepTRG(Cinelli etal.,2014).Projectingneuronsareretrogradelylabeledby injec-tionsofNeurobiotinintothepTRG.Preliminaryresultsshowthat theseneuronsdisplayglutamateimmunoreactivity(unpublished data).Ofnote,bicucullineappliedtothevagalmotoneuronregion duringapneacausedbyionotropicglutamatereceptor blockade doesnotrestoretherespiratoryrhythm,thussuggestingthatthis regiondoesnotpossesstherhythmogenicpropertieshypothesized bypreviousstudies (see e.g. Kawasaki,1979, 1984;Thompson, 1985,1990;Marteletal.,2007).However,thefunctionalroleof GABAergicandglycinergicinputstoneuronslocatedinthevagal motoneuronregionremainsunclear.

6. Respiratoryroleofneurokininsandacetylcholine

Neurokinins(NKs)haveanimportantmodulatoryroleinthe lampreyrespiratorynetwork(Mutoloetal.,2010).Microinjections ofSPaswellasNK1,NK2andNK3receptoragonistsintothepTRG

increasethefrequencyandamplitudeofvagalbursts.Furthermore, SPmicroinjectionsintothepTRG(Fig.6A)restorerhythmic respi-ratoryactivityduringapneainducedbybathapplicationofriluzole andﬂufenamicacidusedtoblocktheburst-promotingcurrents, i.e.thepersistentNa+_current_(I

NaP)andtheCa2+-activated

non-speciﬁccationcurrent(ICAN),respectively.Therhythmogenicrole

of SPis also conﬁrmedby recent ﬁndings (Cinelli et al., 2013) showingthattherespiratoryrhythmcanbere-establishedbySP microinjectedintothepTRGduringapneacausedbyablockade ofionotropicglutamatereceptorswithinthisregion(Fig.6B).The

presenceofanintenseSP-immunoreactivityincloseproximityto pTRGneuronsisconsistentwiththeseﬁndings(Fig.6C).

AChplays animportantexcitatoryrole onrespirationunder basalconditionsandisalsocapableperseofmaintainingrhythmic respiratoryactivitywhenbothfastexcitatoryandinhibitory neuro-transmissionareimpaired.Boththeseeffectsareachievedthrough anactionon␣7nicotinicAChreceptorsofpTRGneurons(Mutolo etal.,2011).Activationof thesereceptorsbynicotineincreases respiratoryfrequency,whiletheirblockadewithD-tubocurarine or␣-bungarotoxinreducesrespiratoryfrequencyandincreasesthe durationofvagalbursts.Combinedhistologicalandfunctional find-ingsstronglysupportthehypothesisthatpTRGneuronsexpressing ␣7nicotinicAChreceptorsmayhavearhythmogenicrole(Mutolo etal.,2011;Cinellietal.,2013).Duringblockadeofbothfast excit-atoryandinhibitoryneurotransmission,therespiratoryrhythmic activitypersists,althoughatreducedfrequency,andissuppressed byblockade of pTRG␣7 nicotinicACh receptors (Fig. 7A). Fur-thermore,duringtheapneainducedbytheblockadeofionotropic glutamatereceptorswithinthepTRG,microinjectionsofnicotine intothesameregionrestorerhythmicrespiratoryactivity(Fig.7B). Itisnoteworthythatimmunohistochemicalexperimentsrevealed thepresenceof␣-bungarotoxinbindingsites(indicatingnicotinic receptors)throughoutthepTRGareaandparticularlyonthesoma ofretrogradelylabeledneuronsprojectingtothevagal motoneu-ronregion(Fig.7C).Inagreementwithpreviousfindings(Pombal etal.,2001;LeRayetal.,2003),wehavealsoprovidedevidence thatcholinergicneuronsareclosetoandintermingledwith retro-gradelylabeledpTRGneurons(Cinellietal.,2013).Together,these findingsidentifyanovelcholinergicmodulatoryandpossibly sub-sidiaryrhythmogenicmechanismwithinthelampreyrespiratory networkand motivatefurtherstudiesontherespiratory roleof cholinergicreceptorsindifferentanimalspecies.

The ﬁndings on the resumption of respiratory rhythm fol-lowing SP or nicotine microinjections into the pTRG ﬁt the “group-pacemaker” hypothesisproposedforrespiratory rhythm generationinmammals(DelNegroetal.,2005;FeldmanandDel Negro,2006).Itwasfoundthatablockadeoftheburst-promoting currentseliminatestherespiratoryrhythmthat,however,could

(5)

Fig.4.RoleofGABAAreceptorsintheresumptionofrespiratoryactivityduringblockadeofionotropicglutamatereceptors.(A)Bathapplicationof20␮MCNQXand

100␮MD-AP5abolishedtherespiratoryrhythmthatwasrestoredby10␮Mbicuculline(Bic)addedtothebath.Aschematicillustrationofadorsalviewofthelamprey mesencephalon/rhombencephalonintheperfusedrecordingchamberisshown.(B)Bilateralmicroinjectionsof1mMBicintothepTRGrestoredtherespiratoryrhythm duringapneacausedbybathapplicationof20␮MCNQXand100␮MD-AP5.ThesiteswhereBicwasmicroinjected(pinkareas)areshownonaschematicillustrationofa dorsalviewofthelampreymesencephalon/rhombencephalon.(C)Suppressionofrespiratoryrhythmicactivity∼1minafteraunilateralmicroinjectionof0.2mMmuscimol (Mus)intothepTRGregion.(D)PhotomicrographofatransversesectionfromtheisthmicregionatthelevelofthepTRG(leftpanel)showingretrogradelylabeledneurons (green)afterbilateralinjectionsofNeurobiotinintothevagalmotoneuronpools.GABAimmunoreactivityisshowninred(scalebar200␮m).Aphotomicrographatahigher magniﬁcationoftheportionofthetransversesection(whitebox)showingretrogradelylabeledneuronswithinthepTRGsurroundedbyGABA-immunoreactivestructuresis reportedintherightpanel(scalebar25␮),ARRN,anteriorrhombencephalicreticularnucleus;I1,isthmicMüllercell;pTRG,paratrigeminalrespiratorygroup;V,trigeminal

motornucleus;VII,facialmotornucleus;IX,glossopharyngealmotornucleus;X,vagalmotornucleus;VA,rawvagalnerveactivity;IVA,integratedvagalnerveactivity. ModiﬁedfromCinellietal.(2014).

berestoredbyincreasingnetworkexcitabilitybyexogenous excit-atoryagents(DelNegroetal.,2005;FeldmanandDelNegro,2006). Theresumptionofrespiratoryrhythmicactivitywassuggestedto resultfromsynapticglutamatergicinterconnectionsthatcombine withtheintrinsic membraneproperties ofneuronswithoutthe involvementofpacemakerneurons.However,howthiscouldhave occurredinourpreparationsduringionotropicglutamatereceptor blockadewithinthepTRGisatpresentonlymatterofspeculation andthereasonsunderlyingrespiratoryrhythmresumptioninthe lampreyremainunclear(seeCinellietal.,2013).Aninvolvementof

metabotropicglutamatereceptorsseemsunlikelysincetheir block-adeduringaconcomitantremovaloffastsynapticexcitatoryand inhibitorytransmissiondidnotproduceanychangeinrespiration (Mutoloetal.,2011).SPeffectsmaybeduetoanincreaseinan ICAN-dependentburstingmechanism(Pe ˜naandRamirez,2004;Ben

MabroukandTryba,2010).Ontheotherhand,nicotinecouldhave producedtheresumptionof rhythmicactivitybyincreasingthe excitabilityofpTRGneuronsthroughaCa2+_-dependent_mechanism

(Albuquerqueetal.,2009).Finally,wecanhypothesizearoleofgap junctionsinrespiratoryrhythmresumption,althoughatpresent

(6)

Fig.5. RespiratoryroleofGABAAandglycinereceptorswithinthevagalmotornucleus.(A)Markedincreasesinrespiratoryfrequency∼4minafteraunilateralmicroinjection

of1mMbicuculline(Bic)intotheregionofvagalmotoneurons(MNregion).(B)Markedincreasesinrespiratoryfrequency∼3minafteraunilateralmicroinjectionof1mM strychnine(Stryc)intothevagalMNregion.SiteswhereBicorStrycmicroinjections(bluearea)wereperformedareshownonaschematicillustrationofadorsalviewof thelampreymesencephalon/rhombencephalon.V,trigeminalmotornucleus;VII,facialmotornucleus;IX,glossopharyngealmotornucleus;X,vagalmotornucleus;VA,raw vagalnerveactivity;IVA,integratedvagalnerveactivity.ModiﬁedfromCinellietal.(2014).

noinformationisavailableontheirpresenceandfunctioninthe lampreyrespiratorynetwork.

7. Evolutionaryconservedcharacteristicsoftherespiratory CPG

Themostimportant findings ontheconnectivity withinthe respiratorynetworkofthelampreyandrelevantneurotransmitter influencesareschematicallyillustratedinFig.8.Webelievethatthe pTRGhasacrucialroleinrespiratoryrhythmgenerationsimilarto thatattributedtothepreBötCinmammals(FeldmanandDelNegro, 2006;Smithetal.,1991).LikethepTRG,thepreBötCcontains pre-dominantlyglutamatergicneuronsthatexpressNK1receptorsand arespecificallysensitivetoopioidsandSP(Grayetal.,1999,2001; Guyenetetal.,2002;FeldmanandDelNegro,2006;Feldmanetal., 2013).ApossibledifferenceisthatthelampreypTRGislocated intherostralrhombencephalon/isthmicregioncorrespondingto therostralpons,whilethepreBötCislocatedinthemedulla.The pontinerespiratorygroupandespeciallytheKölliker-Fusenucleus haveimportant respiratory functions,suchas theregulation of theinspiratory–expiratoryphasetransitionandthegenesisofthe postinspiratoryactivity(DutschmannandDick,2012;Bautistaand Dutschmann,2014;Poonand Song,2014alsoforfurtherRefs.). However,availabledatadonotsupportthenotionthattheydisplay functionalcharacteristicssimilartothoseobservedinthepTRGand inthepreBötC.

Astotheinhibitorycontrolofrespiration,therespiratory rhyth-mogenesispersistsafterablockadeofsynapticinhibition,notonly inneonatalrodentpreparations(reviewedinFeldmanetal.,2013), butalsointheadultlamprey(Rovainen,1983;Bongiannietal., 2006;Cinelli etal.,2014)andintheadultturtle(Johnsonetal., 2002,2007).Inaddition,ablockadeofsynapticinhibitioninthe pre-metamorphictadpoleabolishesﬁctivegillventilation,butnot lungventilation(Galanteetal.,1996;Brochetal.,2002).These ﬁnd-ingsareconsistent,atleasttosomeextent,withrecentresultsin adultmammals(e.g.Bongiannietal.,2010;Feldmanetal.,2013; Janczewskietal.,2013;Kametal.,2013alsoforfurtherRefs.).

Intriguingproposalsonthehomology betweenoscillators in mammalsandlowervertebrateshavebeenadvanceddespitethe

insufﬁciencyof available supporting data(Wilsonet al., 2006). InagreementwithKinkead(2009),webelievethatthelamprey pTRGdisplays a highhomology not onlywith themammalian preBötC,butalsowiththeneuralmechanismsgeneratinglung ven-tilationinamphibians(Wilsonetal.,2002;Vasilakosetal.,2005; ChenandHedrick,2008;Kotticketal.,2013)andturtles(Johnson etal.,2002,2007),ratherthanwiththosethatgenerategill res-pirationintadpoles(Galanteetal.,1996;Brochetal.,2002).All thesedifferentoscillators haveasa possibleunderlyingrhythm generatingmechanismthe“group-pacemaker”model(DelNegro etal.,2005;FeldmanandDelNegro,2006).Inaddition,theydisplay opioidsensitivityand,atleastinfrogsandmammals,SP sensitiv-ity.Admittedly,inthelampreytheactivephaseisexpiration,thus thepTRGcouldmoreappropriatelycorrespondtothe retrotrape-zoid nucleus/parafacial respiratory group, i.e. the hypothesized rostralexpiratoryoscillatorofmammals(seee.g.Onimaruetal., 2009; Thoby-Brisson et al., 2009; Guyenet and Mulkey, 2010; Feldmanetal.,2013;Smithetal.,2013).Thisoscillatormaydisplay burstactivityinvolvingendogenousINaP-dependentproperties,as

itoccursinthepreBötC(FortinandThoby-Brisson,2009; Thoby-Brissonetal.,2009;Molkovetal.,2010).Inaddition,itcontains glutamatergicneuronsthatexpressNK1receptors,butitisnot sen-sitivetoopioids(Mulkeyetal.,2004;Onimaruetal.,2008;Takakura etal.,2008;Lazarenkoetal.,2009;Thoby-Brissonetal.,2009;for reviews,seeFeldmanetal.,2013;GuyenetandMulkey,2010).In conclusion,similarlytootherneurophysiologicalfeatures(Ericsson etal.,2011,2013;Stephenson-Jonesetal.,2011,2012a,2012b),the basicoscillatoryandneuromodulatorymechanismsofthe respira-torynetworkseemtobehighlyevolutionaryconservedregardless oftheirlocationandtheirinspiratoryorexpiratoryfunction. 8. Considerationsontheevolutionarytrendsinrespiratory rhythmgenerationacrossthevertebrateclasses

Theﬁndingthattherespiratoryrhythmgeneratorinthe lam-prey, and possibly alsoin jawedﬁshes, is localized withinthe reticularformationclosetothetrigeminalnucleusisnot surpris-ing.Infact,theevolutionaryoriginofrespiratorymechanismsin vertebratesisfromstructuresandpumpsinitiallyassociatedwith

(7)

Fig.6.RhythmogenicroleofsubstanceP.(A)Bathcoapplicationofriluzole(RIL)andﬂufenamicacid(FFA)at50␮Mabolishedtherespiratoryrhythmthatwasrestarted bybilateralmicroinjectionsof1␮MsubstanceP(SP)intothepTRG.(B)Bilateralmicroinjectionsof1mMCNQXand5mMD-AP5intothepTRGabolishedtherespiratory rhythmthatwasrestored∼1minfollowingbilateralmicroinjectionsof1␮MSPintothesamesites.(C)SPimmunoreactivitywithinthepTRG.Confocalphotomicrographs showingretrogradelylabeledneurons(greensignal)afterinjectionsofNeurobiotinintothevagalmotoneuronpool,SPimmunoreactivity(redsignal)andmergedimage. Scalebar,30␮m.VA,rawvagalnerveactivity;IVA,integratedvagalnerveactivity.AdaptedfromCinellietal.(2013).

feeding(Rovainen,1996;Kardong,2006;Kinkead,2009;Milsom, 2010;Tayloretal.,2010).Atthisstage,thetrigeminalmotor mecha-nismplaysaprominentroleandistheﬁrstmoverintherespiratory sequencethatalsoinvolvesothercranialmotornuclei.Itismainly responsibleforvelarpumpinginthelarvallampreyandforbuccal pumpinginjawedﬁshes(seee.g.Tayloretal.,2010).Inaddition,the velumoflarvallampreysintheadultbecomesavalvethatallows breathingwhenthemouthisengagedinfeedingbehavior(Kinkead, 2009).

Despitethefactthatrespiratoryactivityisproducedbyatidal pump(adultlampreys)orbyasuction/forcepumpdrivenby mus-clesinnervatedbybranchiomericandhypobranchialnerves(jawed ﬁshes),theoriginalgeneratoroftherespiratoryrhythmmayreside inthereticularformationclosetothetrigeminalnucleusandsend driveprojectionstofacial,glossopharyngealandvagal motoneu-rons, thusmaintaininga hierarchically dominant role. Froman evolutionarypointofview,weshouldrecallthatinair-breathing ﬁshes,amphibians,reptiles,birdsand mammalstherespiratory activitychangesprogressivelyfromabuccal/branchialventilation

toaventilationprimarilydrivenbyanaspirationpump(Kinkead, 2009;Milsom,2010;Tayloretal.,2010).Despitethedifferences displayed by the different species in the pattern of conveying air and in the function of the respiratory muscles, evidence is accumulatingthat therespiratory rhythm generatorwithin the reticularformationhasshiftedfromapositionclosetothe trigemi-nalnucleus,thathaslostitsprimarypumpingrespiratoryfunction, toa locationclosetotheothercranialmotor nuclei.These lat-ter, along with spinal motor nuclei innervating the intercostal musclesanddiaphragm,progressivelyacquireaprominent respi-ratoryrole.ThesechangesinthelocationoftherespiratoryCPG obviouslyimplyacaudalmigrationoftheoriginalrhythm generat-ingmechanismorthedevelopmentofanewrespiratoryoscillator ormultipleoscillators,entrainedtoalargedegree(Wilsonetal., 2002,2006;Vasilakosetal.,2005;Tayloretal.,2010;Kotticketal., 2013).Therespiratory CPGofhigher vertebratesand mammals remainsplacedinacranialstrategicpositiontodriveclose brain-stemmotoneuronsthathavetobeengagedinadvanceandtosend excitatory projections to lower respiratory muscles innervated

(8)

Fig.7.Rhythmogenicroleofacetylcholine.(A)Bathapplicationofacocktailsolutioncontaining20␮MCNQX,100␮MD-AP5,10␮Mbicucullineand10␮Mstrychnine depressedrespiratoryactivitythatwascompletelyabolished∼5minafterbilateralmicroinjectionsof2.5␮M␣-bungarotoxin(␣BgTx)intothepTRG.(B)Bilateral microin-jectionsof1mMCNQXand5mMD-AP5intothepTRGabolishedtherespiratoryrhythmthatwasrestored∼1minfollowingbilateralmicroinjectionsof1mMnicotine (Nic)intothesamesites.(C)Distributionof␣-bungarotoxinbindingsitesinthepTRGarea.Photomicrographsshowingretrogradelylabeledneurons(redsignal)following injectionsofTexasRedconjugateddextranintotheregionofvagalmotoneurons,␣-bungarotoxinbindingsites(greensignal)andmergedimage.Retrogradelylabeled neurons(whitearrows)aresurroundedby␣-bungarotoxinbindingsites.Scalebar,25␮m.VA,rawvagalnerveactivity;IVA,integratedvagalnerveactivity.Adaptedfrom

Cinellietal.(2013).

byspinal motoneurons.Infact,brainstemmotoneuronsarestill recruited during respiration, but their main role changed and became that of maintainingstability and patency ofthe upper airways(e.g.VonEuler,1986).Interestingly,trigeminal motoneu-ronsdisplayrhythmicrespiratoryactivityinnewbornrodents(e.g. Jacquinetal.,1999;Koizumietal.,1999,2002)andeveninhumans especiallyunderparticularconditions(Sauerlandetal.,1981; St-Johnand Bledsoe, 1985; Hollowell and Suratt, 1989;Hollowell etal.,1991).Theseﬁndingsmaypossiblyaccountforthe persis-tenceofvestigesoftheoriginaltrigeminaloscillatorandforthe highhomologybetweenthepTRGandthepreBötC.Itshouldbe keptinmindthatwithinthebrainstemandspinalcordneural cir-cuitscapableofgeneratingrhythmicmotorbehaviorsdevelopina segmentalfashion(seeKinkead,2009;Tayloretal.,2010).Each majorgroupofrespiratory motoneuronshasbeensuggestedto becoupledtoitsownrhythmgenerator(ChampagnatandFortin, 1997).Thissegmentalconﬁgurationappearstobetransientand reorganizedoverthecourseofthedevelopmenttoproduce coor-dinatedandeffectivemovements(Kinkead,2009).However,there isevidenceofaresumptionofatrigeminalrhythmfollowing tran-sectionofthebrainstemattheponto-medullaryjunctionaswell

asafterkainicacidlesionsof thedorsal andventralrespiratory groups(St-JohnandBledsoe,1985).Thiscouldsuggestthe pres-ence of a trigeminal oscillator in mammals under appropriate conditions.

Inouropinion,duringtheevolutivestepstowardsmammalian respiration there is a concomitant maturation of the respira-tory network. The primordial respiratory trigeminal oscillator capableof generatinga very simple respiratory pattern is pro-gressivelyembeddedintoacomplexdistributedneuralnetwork subservingthegenerationofthebreathingpatterninmammals (Smithet al.,2007,2013).Webelievethatthemainconcernin the evolution of the neural control of breathing is not repre-sented by thecomplexity of thebreathing pattern that canbe fairlycomplexalsoinlowervertebrates,butbyotherproperties ofrespiration,suchasrhythmstabilization,optimizationofthe energetic cost,integrationwithothernon respiratory functions of respiratory muscles and adjustments to different behavioral and environmental conditions (Von Euler, 1986). Most of the presentedconsiderationsarespeculative,neverthelesstheymay provide hints for further studies not only onthe evolutionary trendsinrespiratoryrhythmgeneration,butalsoonthecontrolof

(9)

Fig.8.Schematicdrawingrepresentingfindingsontheconnectivitywithinthe respiratorynetworkandrelevantneurotransmitterinfluences.ThepTRGregionis shownwithitsprojections(pink)toipsilateralandcontralateralvagal motoneu-ron(red)regionsandtothecontralateralpTRG(Gariépyetal.,2012;Cinellietal., 2013,2014).Excitatory(yellow)andinhibitory(blue)influencesonthepTRGregion (Mutoloetal.,2007,2010,2011;Cinellietal.,2013,2014)andthevagal motoneu-ronregionareillustrated.Glutamatergic(Glu)projectionstothepTRG(green)from neuronslocatedinthevagalareahavealsobeenreported.ACh,acetylcholine;GABA, ␥-aminobutyricacid;Gly,glycine;pTRG,paratrigeminalrespiratorygroupregion; SP,substanceP;X,vagalmotoneuronregion.ModifiedfromCinellietal.(2014).

breathinginmammalsbothunderphysiologicalandpathological conditions.

Acknowledgments

ThisstudywassupportedbygrantsfromtheMinistryof Educa-tion,University,andResearchofItalyandtheA.MenariniUnited Pharmaceutical Industries. E.C. is supported by a Postdoctoral FellowshipfromRegioneToscanaandMenariniUnited Pharma-ceuticalIndustries.

References

Albuquerque,E.X.,Pereira,E.F.,Alkondon,M.,Rogers, S.W.,2009. Mammalian nicotinicacetylcholinereceptors:fromstructuretofunction.Physiol.Rev.89, 73–120.

Bautista,T.G.,Dutschmann,M.,2014.InhibitionofthepontineKolliker-Fusenucleus abolisheseupneicinspiratoryhypoglossalmotordischargeinrat.Neuroscience 267,22–29.

BenMabrouk,F.,Tryba,A.K.,2010.SubstancePmodulationofTRPC3/7channels improvesrespiratoryrhythmregularityandICAN-dependentpacemaker activ-ity.Eur.J.Neurosci.31,1219–1232.

Bongianni,F.,Deliagina,T.G.,Grillner,S.,1999.Roleofglutamatereceptorsubtypes inthelampreyrespiratorynetwork.BrainRes.826,298–302.

Bongianni,F.,Mutolo,D.,Carﬁ,M.,Pantaleo,T.,2002.GroupIandIImetabotropic glu-tamatereceptorsmodulaterespiratoryactivityinthelamprey.Eur.J.Neurosci. 16,454–460.

Bongianni,F.,Mutolo,D.,Cinelli,E.,Pantaleo,T.,2008.Neurokininreceptor modu-lationofrespiratoryactivityintherabbit.Eur.J.Neurosci.27,3233–3243.

Bongianni,F.,Mutolo,D.,Cinelli,E.,Pantaleo,T.,2010.Respiratoryresponsesinduced byblockadesofGABAandglycinereceptorswithintheBötzingercomplexand thepre-Bötzingercomplexoftherabbit.BrainRes.1344,134–147.

Bongianni,F.,Mutolo,D.,Nardone,F.,Pantaleo,T.,2006.GABAergicand glyciner-gicinhibitorymechanismsinthelampreyrespiratorycontrol.BrainRes.1090, 134–145.

Broch,L.,Morales,R.D., Sandoval,A.V.,Hedrick, M.S.,2002. Regulationofthe respiratorycentralpatterngeneratorbychloride-dependentinhibitionduring developmentinthebullfrog(Ranacatesbeiana).J.Exp.Biol.205,1161–1169.

Champagnat,J.,Fortin,G.,1997.Primordialrespiratory-likerhythmgenerationin thevertebrateembryo.TrendsNeurosci.20,119–124.

Chen,A.K.,Hedrick,M.S.,2008.RoleofglutamateandsubstancePinthe amphib-ianrespiratorynetworkduringdevelopment.Respir.Physiol.Neurobiol.162, 24–31.

Cinelli,E.,Mutolo,D.,Robertson,B.,Grillner,S.,Contini,M.,Pantaleo,T.,Bongianni, F.,2014.GABAergicandglycinergicinputsmodulaterhythmogenicmechanisms inthelampreyrespiratorynetwork.J.Physiol.592,1823–1838.

Cinelli,E.,Robertson,B.,Mutolo,D.,Grillner,S.,Pantaleo,T.,Bongianni,F.,2013. Neu-ronalmechanismsofrespiratorypatterngenerationareevolutionaryconserved. J.Neurosci.33,9104–9112.

DelNegro,C.A.,Morgado-Valle,C.,Hayes,J.A.,Mackay,D.D.,Pace,R.W.,Crowder,E.A., Feldman,J.L.,2005.Sodiumandcalciumcurrent-mediatedpacemakerneurons andrespiratoryrhythmgeneration.J.Neurosci.25,446–453.

Dutschmann,M.,Dick,T.E.,2012.Pontinemechanismsofrespiratorycontrol.Compr. Physiol.2,2443–2469.

Ericsson,J.,Silberberg,G.,Robertson,B.,Wikström,M.A.,Grillner,S.,2011. Stri-atalcellularpropertiesconservedfromlampreystomammals.J.Physiol.589, 2979–2992.

Ericsson,J.,Stephenson-Jones,M.,Kardamakis,A.,Robertson,B.,Silberberg,G., Grillner,S.,2013.Evolutionarilyconserveddifferencesinpallialandthalamic short-termsynapticplasticityinstriatum.J.Physiol.591,859–874.

Feldman,J.L.,DelNegro,C.A.,2006.Lookingforinspiration:newperspectiveson respiratoryrhythm.Nat.Rev.Neurosci.7,232–242.

Feldman,J.L.,DelNegro,C.A.,Gray,P.A.,2013.Understandingtherhythmof breath-ing:sonear,yetsofar.Annu.Rev.Physiol.75,423–452.

Fortin,G.,Thoby-Brisson,M.,2009.Embryonicemergenceoftherespiratoryrhythm generator.Respir.Physiol.Neurobiol.168,86–91.

Galante,R.J.,Kubin,L.,Fishman,A.P.,Pack,A.I.,1996.Roleofchloride-mediated inhi-bitioninrespiratoryrhythmogenesisinaninvitrobrainstemoftadpole,Rana catesbeiana.J.Physiol.492,545–558.

Gariépy,J.F.,Missaghi,K.,Chartre,S.,Robert,M.,Auclair,F.,Dubuc,R.,2012.Bilateral connectivityinthebrainstemrespiratorynetworksoflampreys.J.Comp.Neurol. 520,1442–1456.

Gray,P.A.,Janczewski,W.A.,Mellen,N.,McCrimmon,D.R.,Feldman,J.L.,2001. Nor-malbreathingrequirespreBötzingercomplexneurokinin-1receptor-expressing neurons.Nat.Neurosci.4,927–930.

Gray,P.A.,Rekling,J.C.,Bocchiaro,C.M.,Feldman,J.L.,1999.Modulationofrespiratory frequencybypeptidergicinputtorhythmogenicneuronsinthepreBötzinger complex.Science286,1566–1568.

Grillner,S.,2003.Themotorinfrastructure:fromionchannelstoneuronalnetworks. Nat.Rev.Neurosci.4,573–586.

Grillner,S.,2006.Biologicalpatterngeneration:thecellularandcomputationallogic ofnetworksinmotion.Neuron52,751–766.

Guimond,J.C.,Auclair,F.,Lund,J.P.,Dubuc,R.,2003.Anatomicaland physiolog-icalstudyofrespiratorymotorinnervationinlampreys.Neuroscience122, 259–266.

Guyenet,P.G.,Mulkey,D.K.,2010.Retrotrapezoidnucleusandparafacialrespiratory group.Respir.Physiol.Neurobiol.173,244–255.

Guyenet, P.G.,Sevigny,C.P., Weston,M.C., Stornetta,R.L., 2002. Neurokinin-1 receptor-expressingcellsoftheventralrespiratorygrouparefunctionally het-erogeneousandpredominantlyglutamatergic.J.Neurosci.22,3806–3816.

Hollowell,D.E.,Bhandary,P.R.,Funsten,A.W.,Suratt,P.M.,1991.Respiratory-related recruitmentofthemasseter:responsetohypercapniaandloading.J.Appl. Physiol.70,2508–2513.

Hollowell,D.E.,Suratt,P.M.,1989.Activationofmassetermuscleswithinspiratory resistanceloading.J.Appl.Physiol.67,270–275.

Jacquin,T.D.,Sadoc,G.,Borday,V.,Champagnat,J.,1999.Pontineandmedullary con-troloftherespiratoryactivityinthetrigeminalandfacialnervesofthenewborn mouse:aninvitrostudy.Eur.J.Neurosci.11,213–222.

Janczewski,W.A.,Tashima,A.,Hsu,P.,Cui,Y.,Feldman,J.L.,2013.Roleofinhibition inrespiratorypatterngeneration.J.Neurosci.33,5454–5465.

Johnson,S.M.,Wiegel,L.M.,Majewski,D.J.,2007.Arepacemakerpropertiesrequired forrespiratoryrhythmgenerationinadultturtlebrainstemsinvitro?Am.J. Physiol.Regul.Integr.Comp.Physiol.293,R901–R910.

Johnson,S.M.,Wilkerson,J.E.,Wenninger,M.R.,Henderson,D.R.,Mitchell,G.S.,2002.

Roleofsynapticinhibitioninturtlerespiratoryrhythmgeneration.J.Physiol. 544,253–265.

Kam,K.,Worrell,J.W.,Janczewski,W.A.,Cui,Y.,Feldman,J.L.,2013.Distinct inspira-toryrhythmandpatterngeneratingmechanismsinthepreBotzingercomplex. J.Neurosci.33,9235–9245.

Kardong,K.V.,2006.Vertebrates:ComparativeAnatomy,FunctionandEvolution. McGraw-Hill,NewYork.

Kawasaki,R.,1979.Breathingrhythm-generationintheadultlamprey,Entosphenus japonicus.Jpn.J.Physiol.29,327–338.

Kawasaki,R.,1984.Breathingrhythm-generationmechanismintheadultlamprey (Lampetrajaponica).Jpn.J.Physiol.34,319–335.

Kinkead,R.,2009.Phylogenetictrendsinrespiratoryrhythmogenesis:insightsfrom ectothermicvertebrates.Respir.Physiol.Neurobiol.168,39–48.

Koizumi,H.,Ishihama,K.,Nomura,K.,Yamanishi,T.,Kogo,M.,Matsuya,T.,2002.

Differentialdischargepatternsofrhythmicalactivityintrigeminal motoneu-ronsduringﬁctivemasticationandrespirationinvitro.BrainRes.Bull.58, 129–133.

(10)

26 F.Bongiannietal./RespiratoryPhysiology&Neurobiology224(2016)17–26 Koizumi,H.,Nomura,K.,Ishihama,K.,Kogo,M.,Matsuya,T.,1999.Temporal

pat-ternsoftrigeminalrespiratoryactivityinratbrainstem–spinalcordinvitro. Neuroreport10,2609–2613.

Kottick,A.,Baghdadwala,M.I.,Ferguson,E.V.,Wilson,R.J.,2013.Transmissionofthe respiratoryrhythmtotrigeminalandhypoglossalmotorneuronsinthe Ameri-canBullfrog(Lithobatescatesbeiana).Respir.Physiol.Neurobiol.188,180–191.

Kumar,S.,Hedges,S.B.,1998.Amoleculartimescaleforvertebrateevolution.Nature 392,917–920.

Lazarenko,R.M.,Milner,T.A.,Depuy,S.D.,Stornetta,R.L.,West,G.H.,Kievits,J.A., Bayliss,D.A.,Guyenet,P.G.,2009.Acidsensitivityandultrastructureofthe retro-trapezoidnucleusinPhox2b-EGFPtransgenicmice.J.Comp.Neurol.517,69–86.

LeRay,D.,Brocard,F.,Bourcier-Lucas,C.,Auclair,F.,Lafaille,P.,Dubuc,R.,2003.

Nicotinicactivationofreticulospinalcellsinvolvedinthecontrolofswimming inlampreys.Eur.J.Neurosci.17,137–148.

Martel,B.,Guimond,J.C.,Gariepy,J.F.,Gravel,J.,Auclair,F.,Kolta,A.,Lund,J.P.,Dubuc, R.,2007.Respiratoryrhythmsgeneratedinthelampreyrhombencephalon. Neu-roscience148,279–293.

Milsom,W.K.,2010.Adaptivetrendsinrespiratorycontrol:acomparative perspec-tive.Am.J.Physiol.Regul.Integr.Comp.Physiol.299,R1–R10.

Molkov,Y.I.,Abdala,A.P.,Bacak,B.J.,Smith,J.C.,Paton,J.F.,Rybak,I.A.,2010. Late-expiratoryactivity:emergenceandinteractionswiththerespiratoryCpG.J. Neurophysiol.104,2713–2729.

Mulkey,D.K.,Stornetta,R.L.,Weston,M.C.,Simmons,J.R.,Parker,A.,Bayliss,D.A., Guyenet,P.G.,2004.Respiratorycontrolbyventralsurfacechemoreceptor neu-ronsinrats.Nat.Neurosci.7,1360–1369.

Mutolo,D.,Bongianni,F.,Cinelli,E.,Pantaleo,T.,2010.Roleofneurokininreceptors andionicmechanismswithintherespiratorynetworkofthelamprey. Neuro-science169,1136–1149.

Mutolo,D.,Bongianni,F.,Einum,J.,Dubuc,R.,Pantaleo,T.,2007.Opioid-induced depressioninthelampreyrespiratorynetwork.Neuroscience150,720–729.

Mutolo,D.,Cinelli,E.,Bongianni,F.,Pantaleo,T.,2011.Identiﬁcationofa choliner-gicmodulatoryandrhythmogenicmechanismwithinthelampreyrespiratory network.J.Neurosci.31,13323–13332.

Onimaru,H.,Ikeda,K.,Kawakami,K.,2008.CO2-sensitivepreinspiratoryneuronsof

theparafacialrespiratorygroupexpressPhox2bintheneonatalrat.J.Neurosci. 28,12845–12850.

Onimaru,H.,Ikeda,K.,Kawakami,K.,2009.Phox2b,RTN/pFRGneuronsand respi-ratoryrhythmogenesis.Respir.Physiol.Neurobiol.168,13–18.

Pe ˜na,F.,Ramirez,J.M.,2004.SubstanceP-mediatedmodulationofpacemaker prop-ertiesinthemammalianrespiratorynetwork.J.Neurosci.24,7549–7556.

Pombal,M.A.,Marin,O.,Gonzalez,A.,2001.Distributionofcholine acetyltransferase-immunoreactivestructuresinthelampreybrain.J.Comp.Neurol.431,105–126.

Poon,C.S.,Song,G.,2014.Bidirectionalplasticityofpontinepneumotaxic postin-spiratorydrive:implicationforapontomedullaryrespiratorycentralpattern generator.Prog.BrainRes.209,235–254.

Robertson,B.,Auclair,F.,Ménard,A.,Grillner,S.,Dubuc,R.,2007.GABAdistribution inlampreyisphylogeneticallyconserved.J.Comp.Neurol.503,47–63.

Rovainen,C.M.,1977.Neuralcontrolofventilationinthelamprey.Fed.Proc.36, 2386–2389.

Rovainen,C.M.,1979.Neurobiologyoflampreys.Physiol.Rev.59,1007–1077.

Rovainen,C.M.,1983. Generationofrespiratoryactivityby thelampreybrain exposedtopicrotoxinandstrychnine,andweaksynapticinhibitionin motoneu-rons.Neuroscience10,875–882.

Rovainen,C.M.,1985.Respiratoryburstsatthemidlineoftherostralmedullaofthe lamprey.J.Comp.Physiol.A157,303–309.

Rovainen,C.M.,1996.Feedingandbreathinginlampreys.BrainBehav.Evol.48, 297–305.

Russell,D.F.,1986.Respiratorypatterngenerationinadultlampreys(Lampetra ﬂu-viatilis):interneuronsandburstresetting.J.Comp.Physiol.A158,91–102.

Sauerland,E.K.,Orr,W.C.,Hairston,L.E.,1981. EMGpatterns oforopharyngeal musclesduringrespirationinwakefulnessandsleep.Electromyogr.Clin. Neu-rophysiol.21,307–316.

Smith,J.C.,Abdala,A.P.,Borgmann,A.,Rybak, I.A.,Paton, J.F.,2013.Brainstem respiratorynetworks:buildingblocksandmicrocircuits.TrendsNeurosci.36, 152–162.

Smith, J.C., Abdala, A.P., Koizumi, H., Rybak, I.A., Paton, J.F., 2007. Spatial andfunctionalarchitectureofthemammalianbrainstemrespiratory net-work: a hierarchy of three oscillatory mechanisms. J. Neurophysiol. 98, 3370–3387.

Smith,J.C.,Ellenberger,H.H.,Ballanyi,K.,Richter,D.W.,Feldman,J.L.,1991. Pre-Botzingercomplex:abrainstemregionthatmaygeneraterespiratoryrhythm inmammals.Science254,726–729.

St-John,W.M.,Bledsoe,T.A.,1985.Genesisofrhythmicrespiratoryactivityinpons independentofmedulla.J.Appl.Physiol.59,684–690.

Stephenson-Jones,M.,Ericsson,J.,Robertson,B.,Grillner,S.,2012a.Evolutionof thebasalganglia:dualoutputpathwaysconservedthroughoutvertebrate phy-logeny.J.Comp.Neurol.520,2957–2973.

Stephenson-Jones,M.,Floros,O.,Robertson,B.,Grillner,S.,2012b.Evolutionary con-servationofthehabenularnucleiandtheircircuitrycontrollingthedopamine and 5-hydroxytryptophan(5-HT) systems. Proc. Natl.Acad.Sci. USA 109, E164–E173.

Stephenson-Jones,M.,Samuelsson,E.,Ericsson,J.,Robertson,B.,Grillner,S.,2011.

Evolutionaryconservationofthebasalgangliaasacommonvertebrate mecha-nismforactionselection.Curr.Biol.21,1081–1091.

Takakura,A.C.,Moreira,T.S.,Stornetta,R.L.,West,G.H.,Gwilt,J.M.,Guyenet,P.G., 2008.SelectivelesionofretrotrapezoidPhox2b-expressingneuronsraisesthe apnoeicthresholdinrats.J.Physiol.586,2975–2991.

Taylor,E.W.,Leite,C.A.C.,McKenzie,D.J.,Wang,T.,2010.Controlofrespirationin ﬁsh,amphibiansandreptiles.Braz.J.Med.Biol.Res.43,409–424.

Thoby-Brisson,M.,Karlen,M.,Wu,N.,Charnay,P.,Champagnat,J.,Fortin,G.,2009.

Geneticidentiﬁcationofanembryonicparafacialoscillatorcouplingtothe pre-Botzingercomplex.Nat.Neurosci.12,1028–1035.

Thompson,K.J.,1985.Organizationofinputstomotoneuronsduringﬁctive respira-tionintheisolatedlampreybrain.J.Comp.Physiol.A157,291–302.

Thompson,K.J.,1990.Controlofrespiratorymotorpatternbysensoryneuronsin spinalcordoflamprey.J.Comp.Physiol.A166,675–684.

Vasilakos,K.,Wilson,R.J.,Kimura,N.,Remmers,J.E.,2005.Ancientgillandlung oscillatorsmaygeneratetherespiratoryrhythmoffrogsandrats.J.Neurobiol. 62,369–385.

Villar-Cervi ˜no,V.,Barreiro-Iglesias,A.,Fernández-López,B.,Mazan,S.,Rodicio,M.C., Anadón,R.,2012.Glutamatergicneuronalpopulationsinthebrainstemofthe sealamprey,Petromyzonmarinus:aninsituhybridizationand immunocyto-chemicalstudy.J.Comp.Neurol.521,522–557.

VonEuler,C.,1986.Brainstemmechanismsforgenerationandcontrolofbreathing pattern.In:Cherniack,N.S.,Widdicombe,J.G.(Eds.),HandbookofPhysiology, TheRespiratorySystem,ControlofBreathing,II.AmericanPhysiologicalSociety, Bethesda,Maryland,pp.1–67.

Wilson,R.J.,Vasilakos,K.,Harris,M.B.,Straus,C.,Remmers,J.E.,2002.Evidencethat ventilatoryrhythmogenesisinthefroginvolvestwodistinctneuronal oscilla-tors.J.Physiol.540,557–570.

Wilson,R.J.,Vasilakos,K.,Remmers,J.E.,2006.Phylogenyofvertebrate respira-toryrhythmgenerators:theOscillatorHomologyHypothesis.Respir.Physiol. Neurobiol.154,47–60.