Glutamatergic Signaling from the Parabrachial Nucleus Plays a Critical Role in Hypercapnic Arousal

14 Pages·2013·6.67 MB·English

TheJournalofNeuroscience,May1,2013•33(18):7627–7640•7627
Systems/Circuits
Glutamatergic Signaling from the Parabrachial Nucleus Plays
a Critical Role in Hypercapnic Arousal
SatvinderKaur,1NigelP.Pedersen,1ShigefumiYokota,1ElizabethE.Hur,1PatrickM.Fuller,1MichaelLazarus,2
NancyL.Chamberlin,1*andCliffordB.Saper1*
1DepartmentofNeurology,PrograminNeuroscience,andDivisionofSleepMedicine,HarvardMedicalSchool,BethIsraelDeaconessMedicalCenter,
Boston,Massachusetts02215,and2DepartmentofMolecularBehavioralBiology,OsakaBioscienceInstitute,Suita,Osaka565-0874,Japan.
The mechanisms of arousal from apneas during sleep in patients suffering from obstructive sleep apnea are not well understood.
However,weknowthatrespiratorychemosensorypathwaysconvergeontheparabrachialnucleus(PB),whichsendsglutamatergic
projectionstoavarietyofforebrainstructurescriticaltoarousal,includingthebasalforebrain,lateralhypothalamus,midlinethalamus,
andcerebralcortex.WetestedtheroleofglutamatergicsignalinginthispathwaybydevelopingananimalmodelforrepetitiveCO
2
arousals(RCAs)andinvestigatingtheeffectofdeletingthegeneforthevesicularglutamatetransporter2(Vglut2)fromneuronsinthePB.
WeusedmicewithloxPsequencesflankingexon2oftheVglut2gene,inwhichadeno-associatedviralvectorscontaininggenesencoding
CrerecombinaseandgreenfluorescentproteinweremicroinjectedintothePBtopermanentlyandselectivelydisruptVglut2expression
whilelabelingtheaffectedneurons.WerecordedsleepinthesemiceandtheninvestigatedthearousalsduringRCA.Vglut2deletionsthat
includedtheexternallateralandlateralcrescentsubdivisionsofthelateralPBmorethandoubledthelatencytoarousalandresultedin
failuretoarouseby30sin(cid:1)30%oftrials.Bycontrast,deletionsthatinvolvedthemedialPBsubdivisionhadminimaleffectsonarousal
duringhypercapniabutinsteadincreasednon-rapideyemovement(NREM)sleepby(cid:2)43%duringthedarkperiod,andincreaseddelta
powerintheEEGduringNREMsleepby(cid:2)50%.OurresultssuggestthatglutamatergicneuronsinthelateralPBarenecessaryforarousals
fromsleepinresponsetoCO ,whilemedialPBglutamatergicneuronsplayanimportantroleinpromotingspontaneouswaking.
2
Introduction Oneprobleminstudyingsleepapneahasbeenthedifficultyin
Patientswithobstructivesleepapnea(OSA)sufferfromrepeated establishing animal models, particularly in rodents, where the
cyclesduringsleepoflossofupperairwaymuscletone,airway underlyinganatomyandphysiologyofcorticalarousalhasbeen
collapse,andapnea,followedbyarousalwithre-establishmentof carefullystudied.ThearousalsthatoccurduringOSAcorrelate
theairway,thenfallingbacktosleep.Thiscyclemayberepeated onlylooselywiththelevelofhypoxiaexperienced,moreclosely
hundreds of times over the course of the night. Although the withthelevelofhypercapnia,andbestwiththelevelofnegative
arousalsarebrief,andthesleepingindividualmaynotevenno- airwaypressuregeneratedduringattemptstobreathe(Gleesonet
tice them, the subsequent sleep fragmentation and chronic, al., 1990). However, the degree of negative pressure generated
intermittenthypoxiacausedaytimesleepinessandcognitiveim- duringattemptstobreatheisproportionaltothelevelofchemo-
pairment, as well as diabetes and cardiovascular pathology receptorstimulation.Becauseitisdifficulttosimulatenegative
(Loredoetal.,1999;Jain,2007;Kimetal.,2007;Tregearetal., airwaypressureinafreelybehavinganimal,wehaveestablisheda
2009).Despitetheimportanceoftheselife-savingarousalsdur- protocolofrepetitivebriefhypercapnia-elicitedarousalsinmice
ingapnea,theirmechanismsaresurprisinglypoorlydefined. asamodelforthearousalsthatoccurduringOSA.
Recentevidenceimplicatestheparabrachialnucleus(PB)and
theadjacentpreceruleusarea(PC)inpromotingcorticalarousal
ReceivedJan.8,2013;revisedFeb.24,2013;acceptedMarch15,2013. (Fuller et al., 2011). Furthermore, the PB is also a key site for
Authorcontributions:S.K.,N.P.P.,N.L.C.,andC.B.S.designedresearch;S.K.andS.Y.performedresearch;S.K., respiratoryregulation(ChamberlinandSaper,1994;Chamberlin,
S.Y.,E.E.H.,P.M.F.,andM.L.contributedunpublishedreagents/analytictools;S.K.,N.L.C.,andC.B.S.analyzeddata;
2004)andshowsextensivecFosexpressionafterhypercapniaorhyp-
S.K.,N.P.P.,N.L.C.,andC.B.S.wrotethepaper.
ThisworkwassupportedbyNationalInstitutesofHealthGrantP01HL095491andagrantfromtheG.Haroldand oxia(Teppemaetal.,1997;Berquinetal.,2000),particularlyinthe
LeilaY.MathersFoundation.E.E.H.receivedpostdoctoralsupportfromGrantsF32NS063673andT32HL007901.We ventral areas in the lateral PB (LPB) [i.e., in the external lateral
thankQuanHaandMinhHafortheirexcellenttechnicalsupport,andSathyajitBandaruformaintainingthemouse (PBel), lateral crescent (PBlc), and Ko¨lliker-Fuse (KF) subnuclei,
breedingprogram.WearealsoindebtedtoDrs.BradfordLowellandQingchunTong,whoproducedtheVglut2flos/floxand
andintheventrolateralpartofthemedialPB(MPB)].Microstimu-
Vglut2-ires-Cremiceandsupplieduswiththebreederpairs,andwethankDr.RolfSprengelatUniversityofHeidel-
lationstudiesfoundthatactivationofthesesameareascausedhy-
bergfordonatingtheplasmidconstructfortheAAV-iCre-2A-Venus,andtoDr.Dr.Jeng-ShinLeeatHarvardGene
TherapyInitiativeforhelpinpackagingit. perpnea(ChamberlinandSaper,1994).Thisrespiratorycomponent
*N.L.CandC.B.S.contributedequallytothiswork. ofthePBalsoreceivesdenseinputsfromcellgroupsthatconveyCO
2
CorrespondenceshouldbeaddressedtoCliffordB.Saper,MD,PhD,Dept.ofNeurology,BethIsraelDeaconess
chemosensory information, including the nucleus of the solitary
MedicalCenter,330BrooklineAvenue,Boston,MA02215.E-mail:[email protected].
tract (NTS) and retrotrapezoid nucleus (Herbert et al., 1990;
DOI:10.1523/JNEUROSCI.0173-13.2013
Copyright©2013theauthors 0270-6474/13/337627-14$15.00/0 Abbottetal.,2009;Depuyetal.,2011;Songetal.,2011;Bochorishvili
7628•J.Neurosci.,May1,2013•33(18):7627–7640 Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia
etal.,2012),andfromthemedullaryraphe
(serotoninergic chemosensory nuclei)
(Steinbusch,1981;Milleretal.,2011;Bang
etal.,2012).AsthePBprojectsheavilyto
theforebrainarousalsystem,includingthe
hypothalamus, thalamus, basal forebrain,
andcortex(SaperandLoewy,1980;Fulwiler
andSaper, 1984), we hypothesized that
the PB mediates arousal responses to
hypercapnic/hypoxic stimuli via its
forebrainprojections.
MostPBneuronsexpressthevesicular
glutamate transporter type 2 (Vglut2; Figure1. RCAprotocol.A,Mouseintheplethysmographchamber,connectedtocablesforrecordingEEGandEMG.Thischamberis
see the Allen Brain Atlas at http://www. placedonthetopofareceiver,whichacquirestheEKG,temperature,andactivitydata.B,DuringRCA,asolenoidswitchesthegasintake
brain-map.org/)(Niuetal.,2010),raising fromnormocapnicair(O,21%;N,79%)toeitherahypercapnic(CO,10%)orhypoxic-hypercapnic(CO,10%;O,10%)mixture[noterise
2 2 2 2 2
the possibility that the PB arousal path- incarbondioxidepartialpressure(pCO)andfallinoxygenpartialpressure(pO)atarrowsinthisexample].Micespontaneouslysleepand
2 2
waysarelikelytobeglutamatergic.Totest awaken(e.g.,themouseisawakebeforethesecondCO trialsinthisexample).However,onlytrialswherethemouseisclearlyinNREM
2
these hypotheses, we focally deleted the sleepforatleast30sbeforeonsetoftheCO2(e.g.,thefirstandthirdCO2trialinthisexample)areusedtoexaminearousal.Duringthese
trials,thearousals,whichtypicallyoccurwithinafewsecondsafteronsetoftheCO stimulus,arejudgedbyEEGarousal(lossofhigh-
Vglut2genetoeliminateglutamatesignal- 2
amplitudedeltawavesandappearanceoflow-voltagefastEEG),whichisusuallyaccompaniedbyEMGactivation.
ingfromthePB,andexaminetheeffecton
arousalsfromsleepelicitedbyeitherhy-
binase.However,incellsthatexpressCrerecombinase,theCrecleaves
percapniaorhypoxichypercapnia.
offthemCherrysequenceenablingtheexpressionofDTA,whichleadsto
celldeletion.Thisvectorwasdesigned,produced,andvalidatedbyDr.
MaterialsandMethods
MichaelLazarusatOsakaBioscienceInstitute,Japan,andDr.PatrickM.
Animals Fullerinourlaboratory.
We used homozygous Vglut2flox/flox mice (n (cid:3) 42) and homozygous
wild-type(n(cid:3)5)littermates(asdeterminedbytailDNAPCRanalysis)for Surgery
experimentalandcontrolanimals.TheVglut2conditionalknock-outmice TodeletethesecondexonoftheVglut2gene,westereotaxicallymicroin-
wereoriginallypreparedbyTongetal.(Tongetal.,2007)andhaveloxPsites jectedthehomozygousVglut2flox/floxmice(n(cid:3)42)ortheirwild-typelitter-
flankingthesecondexonoftheVglut2gene(Vglut2flox/flox).Theoriginal matesascontrols(n(cid:3)5)withAAV-Creasdescribedabove(volumevarying
129Smicewereback-crossedwithC57BL/6Jmicefor2–4generations. from75to300nl)orwithAAV-GFP(n(cid:3)8).Theinjectionstargeteddiffer-
WealsousedaVglut2-ires-Crelineofmice(n(cid:3)6)inasecondsetof entsubnucleiofthePB[(anteroposterior:(cid:5)5.0to(cid:5)5.3mm;mediolateral:
experiments,whereCre-recombinaseisexpressedincellsthatexpress (cid:7)1.1to(cid:7)1.3mm;dorsoventral:(cid:5)2.5to(cid:5)2.9mm)usingaglassmicropi-
Vglut2; this specificity has been validated by Vong and colleagues by petteandanairpressureinjectionsystem.Thesemicewerealsotheninstru-
crossingthesemicewithgreenfluorescentprotein(GFP)reportermice mentedwithEEGandEMGelectrodesandwereimplantedwithtelemetric
(Vongetal.,2011).Weusedheterozygousmiceinthisstudy(i.e.,Vglut2- transmittersintraperitoneallyfortherecordingofEKG,bodytemperature,
ires-Cre(cid:4)/(cid:5)).Theoriginal129SVglut2-ires-Cremicewerebackcrossed andlocomotoractivity(PhysioTelETA-F10,DataScienceInternational).
withC57BL/6Jmiceatleastfortwogenerations. Inanadditionalexperiment,forselectivedeletionoftheVglut2cellsin
Allmiceusedinexperimentsweremale.Animalsweremaintainedon thePB,Vglut2-Cremice(n(cid:3)6)wereinjectedwithAAV-DTA(volume,
a12hlight/darkcyclewithadlibitumaccesstowaterandfood.Allanimal 150 nl) stereotaxically targeted to the lateral PB. These were also im-
proceduresmetNationalInstitutesofHealthstandards,assetforthinthe plantedwithEEGandEMGelectrodesforrecordingsleep.
GuidefortheCareandUseofLaboratoryAnimals,andallprotocolswere
approvedbytheBethIsraelDeaconessMedicalCenterInstitutionalAn- Dataacquisition
imalCareandUseCommittee. Sevendaysaftersurgery,micewereattachedtotherecordingcablesand
acclimatizedtotherecordingchamberfor2d.Micewerethenrecorded
Vectors
forsleepfora48hperiodusingapreamplifier(PinnacleTechnology)
Theadeno-associatedviralvectors(AAVs,eitherserotype8or10)that
connectedtoadataacquisitionsystem(8200-K1-SE)andSireniaSoft-
weusedcontainedthegeneforCrerecombinase(AAV-Cre,n(cid:3)8),the
ware(bothfromPinnacleTechnology).BasedonlossofVglut2expres-
geneforGFP(AAV-GFP,n(cid:3)8),orboth(AAV-GFP-ires-Cre,n(cid:3)9).In
sion in our preliminary studies, Cre-induced recombination occurs
anadditionalsetofmice(n(cid:3)22),weusedanAAVcontainingboththe
slowlyoveraperiodofseveralweekspostinjectioninthesemice,with
genesforCreandforVenus,aGFP,connectedbyaself-processing2A
minimalchangeat1week,substantialrecombinationby3weeks,and
viral peptide bridge (AAV-iCre-2A-Venus). AAV/rh10.CMV.PICre.RBG maximalrecombinationby(cid:2)5weeks.Soafterapost-injectionperiodof
[serotype10AAV-Cre:titer(cid:3)1.13(cid:6)1013GC/mlwithyieldof7.11(cid:6)1012
5weeks,werecordedsleepinthesemiceagainfor48h(after2dof
genomecopies(GC)/ml]wasacquiredfromtheGeneTherapyProgram
acclimatization).Becausethe1weekpostinjectionanimalswerenotdif-
VectorCoreattheUniversityofPennsylvania.AAV-MCS8-GFP-IRES-
ferentfromcontrolsinwake–sleepbehaviororCO responsiveness(see
iCre(serotype8AAV-GFP-ires-Cre:titer(cid:3)8.1(cid:6)1012GC/ml)waspro- 2
text),wecomparedthe1and5weekrecordingsfromindividualanimals
ducedbyHarvardGeneTherapyInitiative,Boston,byDr.Jeng-ShinLee.
aspaireddata(e.g.,formeasuringEEGdeltapower).
TheplasmidconstructfortheAAV-iCre-2A-Venuswasgenerouslydo-
Followingthesleepstudy,micewerethenacclimatizedtotheplethys-
natedbyDr.RolfSprengelattheUniversityofHeidelberg,Heidelberg,
mographicchamberbeginningat9:00A.M.for3–4honeachtestday,
Germany (Tang et al., 2009). This plasmid was then cloned and se-
andthenreceivedoneofthefollowingprotocolsinarandomorder:
quencedinourlaboratoryandpackagedbytheHarvardGeneTherapy
Initiative,Boston,byDr.Jeng-ShinLeeinAAV(serotype8AAV-iCre- Air:air—switchingbetweentwochannelsofnormocapnicair(artificial
2A-Venus:titer(cid:3)9.4(cid:6)1012GC/ml). air)(21%O ,79%N ).
2 2
Inaddition,wealsousedanAAVconditionallyexpressingsubunitA Air:hypercapnicmixture—switchingbetween300sofnormocapnicair
ofdiphtheriatoxin(AAV-lox-mCherry-lox-DTA-WPRE;orAAV-DTA) followedby30sof10%CO ,21%O ,and69%N .
2 2 2
intheVglut2-Cremice.Inthisvectorthelox-flankedmCherrysequence Air:hypoxic-hypercapnicmixture—switchingbetween300sofnormo-
preventsthetranscriptionofDTAincellsthatdonotexpressCrerecom- capnicairfollowedby30sof10%O ,10%CO ,and80%N .
2 2 2
Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia J.Neurosci.,May1,2013•33(18):7627–7640•7629
Table1.Listofprimaryandsecondaryantibodiesusedforimmunostaining
Primaryantibodyovernightincubationsatroomtemperature
Catalog Secondaryantibody
Antigen Host Dilution Source number Immunogen Specificity (2hincubation) Dilution
GFP Mouse 1:500 Invitrogen A11120 GFPisolatedfrom Nostaininginuninjected Donkeyanti-mouse 1:100
Aequoreavictoria animals alexafluor488
(Invitrogen)
GFP Rabbit 1:5000 Invitrogen A11122 GFPisolatedfrom Nostaininginuninjected Donkeyanti-rabbit 1:500
Aequoreavictoria animals biotinylated
Cre Rabbit 1:10,000 Novagen 69050 BacteriophageP1qa Westernblotshowedbindingto Donkeyanti-rabbit 1:500
Cre-recombinase,35 purifiedCrerecombinase; biotinylated
kDaprotein,native nonedetectedinuninjected
anddenatured mousebrain
Vglut2 Rabbit 1:500 Synaptic 135403 Strep-Tagfusionprotein PreincubationoftheVglut2 Donkeyanti-rabbit 1:100
Systems ofratVglut2(amino antibodywithimmunogen alexafluor546
acids510–582) peptideblockslabeling (Invitrogen)
(Grazianoetal.,2008).
Neu-N Mouse 1:1000 Millipore MAB377 Purifiedcellnucleifrom Stainingonlyofneuronalcell Donkeyanti-rabbit 1:500
mousebrain groupsinbrain,notblood biotinylated
vesselsormeningesorin
whitematter
Acousticstimulation(4kHzrangingfrom2to30dB)—acousticstimuli foreachmouse.Allstatisticalanalysesweredoneusingstatisticalsoft-
rangingfrom2dBto30dBwerepresentedfor10sat300sintervals,with ware(Sigmastat).Aprobabilityof(cid:8)0.05wasconsideredsignificant.
eachtonepresented10–12times,inascendingorderofintensity.Aswiththe Analysisofarousaltohypercapnia.EEGarousalsduringrepetitiveCO
2
CO stimulus,onlythoseepochswherethemicewereasleepfor30sbefore arousals(RCAs)wereanalyzedbyEEGactivation(notetheobviousloss
2
thestimuluswereanalyzedforcalculatingthelatencyofarousaltostimulus. ofdeltainFig.1B),whichisusuallyaccompaniedbyEMGactivation
(Fig.1).Becauseoccasionalspontaneousarousalsalsooccurredbefore
Allrecordingsduringrecurrentstimulationweredoneinaplethysmo-
theCO exposure,weonlyanalyzedthosetrialswhereanimalswerein
graphicchamber(unrestrainedwhole-bodyplethysmograph,BuxcoRe- 2
NREMsleepforatleast30sbeforetheCO stimulus.Thisalsoelimi-
searchSystems)allowingustorecordthebreathingofthemousewhilein 2
natedanytrialsinwhichtheanimalswereinREMsleepduringthe30s
thechamber.Electroencephalogram(EEG)andelectromyogram(EMG)
beforetrialonset.REMsleepperiodstypicallyaremuchshorter(hence
were recorded using Pinnacle preamp cables connected to the analog
wewouldexpecttoseemorefrequentspontaneousawakeningduringthe
adaptor(8242,PinnacleTechnology)(Fig.1).Gaslevelsinthechamber
trials)andthereweretoofewtrialsduringREMsleeptoprovidereliable
werecontinuouslymonitoredusingCO andO monitorsfromCWE,
2 2 statisticalanalysis.WescoredthedurationandlatencyofanyEEGacti-
Inc.EEG,EMG,respiration,andCO andO levelswerefedintoanAxon
2 2 vationthatoccurredafteronsetofRCA.Thetrialsinwhichanimalsdid
Digidata1322Aanalog-to-digitalconverterandthesignalswereacquired
notawakenduringthe30softhestimulusweremarkedasfailureto
using Axoscope software (Molecular Devices). The DSI transmitters
arousetostimulus.Forthe30speriodofthehypercapnicstimulus,the
wereactivatedusingamagneticswitchandthedataacquiredusingData-
relationshipbetweenthelatencyofarousalsandtheminuteventilation
questA.R.T.(DataScienceInternational).
(i.e.,respiratoryrateperminutetimesmeantidalvolume)wastested
Dataanalysis statisticallyusingPearsoncorrelationcoefficients.
Sleepanalysis.Digitizedpolygraphicdatawereanalyzedoff-linein10s Histologicalanalysis.Attheconclusionoftheexperiment,theanimals
binsusingSleepSignsoftware(Kissei).Thesoftwareautoscoredeach were perfused with 0.9% saline followed by 10% buffered formalin.
epochusinganalgorithmthatidentifiedthreebehavioralstatesbasedon Brainswereharvestedforanalysisoftheeffectivelocationoftheinjection
EEGandEMG.Theautoscoreddatawerethencheckedatleasttwicevisually site.Brainswerekeptin30%sucrosefor2dandsectionswerecutat30
formovementandanyotherartifactandtoconfirmorcorrectautomatic (cid:1)musingafreezingmicrotomeinfourseries.
stateclassification;concurrentvideoimagesoftheanimal’sbehavioralso Immunohistochemistry.Oneoftheseriesofsectionswasstainedbyin
aidedinthisprocess.Over-readingofthesleeprecordingsweredoneaccord- situ hybridization for Vglut2 using an exon 2-specific probe (radiola-
ingtopreviouslypublishedcriteria(NeckelmannandUrsin,1993;Kauret beled)(Tongetal.,2007).Asecondseriesofsectionswasstainedfor
al.,2008,2009).Theepochswithmovementartifactswereexcludedfrom Vglut2byinsituhybridization(usingadigoxigenin-labeledVglut2ribo-
subsequentEEGspectralanalysis.EEGdataweredigitallylow-passfilteredat probe)followedbyimmunohistochemistryforeitherCrerecombinase
50Hzandtapered(Hanningwindow),andspectralanalyseswasconducted or GFP. A third series was immunostained through the injection site
usingafastFouriertransform.TheintegratedpoweroftheEEGwasana- either for GFP (mouse anti-GFP, 1:500) or Cre (rabbit anti-Cre,
lyzedin2sbinsandaveragedover12hlight/darkcyclesinthefollowing 1:10,000) and through the forebrain for double immunofluorescence
frequencyranges:delta(0.5–4Hz),theta(4.5–8Hz),alpha(8.5–13Hz), withantibodiesfortheGFP(mouseanti-GFP,1:500)andVglut2(rabbit
beta(13.5–30Hz),andgamma(30.5–50Hz). anti-Vglut2,1:500;Table1).Sectionsforimmunostainingwerefirstin-
The changes in amount of time spent in wake, nonrapid-eye- cubatedin0.1Mphosphatebufferand1%H2O2for5–10minfollowed
movement(NREM)sleep,andrapid-eye-movement(REM)sleepindif- bythreewashingsin0.1Mphosphatebuffer.Inanothersetofexper-
ferent treatment groups and across time (1 week postinjection and 5 iments,Cre-specificablationofneuronswasanalyzedbyimmuno-
weekspostinjection)inexperimentalgroups(AAV-Cre-injected)andin staining with Neu-N (mouse anti-Neu-N, 1:1000), followed by a
control animals (AAV-GFP-injected Vglut2flox/flox mice, AAV-Cre- donkeyanti-mousebiotinylatedsecondaryantibody(1:500).Forall
injected wild-type mice) were compared statistically using two-way theimmunohistochemicalstainingthatinvolvedvisualizationusinga
ANOVAfollowedbyaHolm-Sidakposthoctestformultiplecompari- diaminobenzidine (DAB) reaction, the sections after the overnight
sons.EEGpowervaluesinthedeltafrequencyrange(0.5–4.0Hz)were incubationwithprimaryantiserumwereincubatedintherespective
normalizedaspercentagesofthetotalpowerinthe0.5–50Hzfrequency secondaryantibodies(Table1)for2h,followedbyincubationinABC
rangeandthencomparedwiththeirrespectivecontrolvaluesat1week reagents(1:1000;VectorLaboratories)for90min,thenwashedagain
7630•J.Neurosci.,May1,2013•33(18):7627–7640 Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia
Figure2. PhotomicrographsshowingCre-immunoreactivity(brown)againstaNissl-stainedbackground(blue)intheneuronsinthePBregionafterinjectionofAAV-CreinVglut2flox/floxmice.
ThepresenceofCre-immunoreactivitymarksthenucleiofneuronswithfocaldeletionofVglut2expression.A,BycomparisontosectionsdemonstratingtheNissl-stainedPBsubnuclei,micewithCre
stainingindifferentsubdivisionsofthePBcomplexwerecategorizedintofivegroups.B,InjectionfromtheVLPBgroupshowingCreexpressionthroughoutthelateralPB,includingitsmost
ventrolateralcomponent,theexternallateralandlateralcrescent(el/lc)subnuclei,aswellastheadjacentcentralanddorsallateralsubnuclei(cl,dl).C,TheVLPB(cid:4)KFgroup,inwhichCreexpression
includedthePBel/lcandextendedfurtherrostrallyandventrallyintotheKFsubnucleus,andinthiscaseintotheMPBaswell.TheareamarkedasKFrepresentsthemostcaudalpartoftheKF
subnucleus;mostofthecellgroupisrostraltothislevel.D,ExampleoftheVLPB(cid:4)MPBgroupinwhichmicehadCreexpressionthroughoutthelateralPB,includingthePBel/lcandalsoincludedthe
MPB.E,ExampleofaninjectioninthedlandclPBsubdivisions(DLPBgroup),butexcludingthePBel/lc.F,IntheDLPB(cid:4)MPBgroup,CreexpressionwaspresentthroughoutthemedialPBandalso
includedpartsofthePBdl/cl,butagainexcludedthePBel/lc.G,QuantificationofinvolvementofthePBel/lcindifferentinjectiongroups.ThenumbersofCre-immunoreactive(Cre-ir)neuronsinthe
PBel/lcwerequantified(mean(cid:7)SEM)andcomparedwiththenumbersofNissl-stainedneuronsinthosecellgroups.InthethreegroupsthatincludedthePBel/lc,anaverageof(cid:2)70%ofthe
neuronsinthatsubnucleusexpressedCre,whereasintheDLPBandDLPB(cid:4)MPBgroups,(cid:8)20%did(F (cid:3)45.81;p(cid:8)0.01).scp,superiorcerebellarpeduncle;vl,ventrolateralPBsubnucleus;
(4,22)
vsct,ventralcerebrospinaltract.Scalebar,100(cid:1)m.
andincubatedina0.06%solutionof3,3-diaminobenzidinetetrahy- Insituhybridization.WecombinedinsituhybridizationoftheVglut2
drochloride (Sigma-Aldrich) and 0.05% CoCl and 0.01% NiSO neuronsusinganonradioactiveprobeforVglut2(digoxigenin-labeled
2 4
(NH )inPBSplus0.02%H O for5min.Finally,thesectionswere Vglut2riboprobescorrespondingtoexon2(nucleicacids830–1287),
4 2 2
mountedonslides,dehydrated,cleared,andcoverslipped.Sections whichwasbetweentheloxPsitesintheVglut2flox/floxmice(Tongetal.,
for double-immunofluorescence staining for both GFP and Vglut2 2007)withimmunohistochemistryfortheCre/GFPlabelingusingthe
wereincubatedinfluorescent-labeledsecondaryantibodies(Table1) rabbitanti-Cre(1:5000)antibodyorrabbitanti-GFP,inoneseriesof30
for 2 h and coverslipped with fluorescence mounting media (Dako (cid:1)msections(1:5000)(Table1).Sectionswereincubatedovernightinthe
NorthAmerica). hybridization buffer containing 5 (cid:1)g/ml Vglut2 riboprobe at 60°C.
Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia J.Neurosci.,May1,2013•33(18):7627–7640•7631
4-toludinesalt(asubstrateforalkalinephos-
phatase)for6–7hatroomtemperature.Re-
peatedwashingswithTBSresultedinspecific
labelingoftheVglut2-positiveneuronsbythe
purple reaction product. Next, the sections
weretreatedwithrabbitanti-Creantibody(1:
5000) overnight at room temperature, and
then reacted with the biotinylated secondary
antibodytorabbitIgG.TheCrewasvisualized
withtheavidin-biotincomplex(1:1000;Vector
Laboratories)andthestandardDABreaction
(seeabove).Finally,sectionsweremountedon
slides,dehydrated,cleared,andcoverslipped.
Analysisofinjectionspread.Eachinjection
ofAAV-Cretransfectedauniquepatternof
neurons in the PB. The spread of injection
dependedonthenaturaldiffusionbarriersin
thetissue(mainlyfibertracts).Wetherefore
divided our cases into groups for analysis
basedonwhetherornottheyincludedlarge
numbersofneuronsintherespiratoryPBas
described earlier (Chamberlin and Saper,
1994).ThebulkoftherespiratoryPBismade
upoftheexternallateralsubnucleus(PBel)
andtheadjacentlateralcrescentsubnucleus
(PBlc).Theborderbetweenthesetwonuclei
isessentiallyindistinguishableinmicewith-
outretrogradelabeling(thePBelprojectsto
theamygdalaandthePBlctothemedulla),
sotheywerecountedasasinglestructurein
ouranalysis(Fig.2A,el/lc).TheKFsubnu-
cleusinmiceisverysmallandlocatedonlyat
farrostralPBlevels.Whileasmallnumberof
MPB neurons are in the area that receives
respiratory afferents, it is difficult to delin-
eate this region accurately. Hence, for the
purposeofquantifyingtheinvolvementofthe
respiratoryPBbyinjections,wecountedthe
numbers of Cre(cid:4) neurons in the PBel/lc
(Fig.2G).Thiscellgroupiseasilyidentified
onNissl-stainedsections.So,ontwosections
((cid:2)90–100(cid:1)mapart)throughthefarrostral
PB and middle PB, we counted the Nissl-
Figure3. DemonstrationofVglut2mRNAdeletioninneuronstransfectedwithCrerecombinase.A,B,Mergedimagesshowing stainedneuronsinthenucleus,aswellasthe
theinjectionofAAV-GFPinthelateralPBofacontrolmouse(A)andAAV-GFP-ires-CreinaVglut2flox/floxmouse(B).Thesesections neurons with Cre-immunoreactive nuclei.
werelabeledforGFPimmunoreactivity(green)andforVglut2mRNA(red)byinsituhybridization.Theinsetsshowcellsatthewhite The diameters of 20 nuclei of each type
arrowheadsathighermagnification.Inthecontrolmouse,nearlyalloftheGFP(cid:4)neuronsalsolabelforVglut2mRNA(yellow). (Cre(cid:4)andCre(cid:5))peranimalwerethenmea-
However,inB,thereisnodouble-labeling,confirmingthatVglut2expressionhasbeendeletedfromtheGFP-expressing(and sured, and Abercrombie’s correction factor
Cre-expressing)neurons.C,D,DeletionofexpressionofVglut2mRNA(purple)inthecytoplasmofneuronsfromVglut2flox/floxmice wasapplied.
thatexpressCre-recombinaseintheirnuclei(brown)afteraninjectionofAAV-GFP-ires-CreintothelateralPB.D,Higher-power
photomicrographoftheboxshowninwhitedashedlinesinC,demonstratingthatpurplecytoplasmindicatingVglut2mRNAis Results
foundonlyinneuronswithaclear(non-Cre-stained)nucleus(openarrow,lowerright),whereasnoneisfoundinthecytoplasmof
InjectionsofAAV-Creproducefocal
theneuronswithbrownCre-stainednuclei(blackarrow).E,F,Vglut2proteindeletionfromaxonterminals.Mergedz-stacksof
lossofVglut2expressioninPB
confocalfluorescenceimagesofasectionimmunostainedforVglut2terminals(red)andGFP(green)inthebasalforebrainafterthe
neuronalcellbodiesandaxonterminals
microinjectionsofAAV-GFP(E)orAAV-GFP-ires-Cre(F)inthePB.Anterogradelylabeledfibersandterminals(green)couldbe
inVglut2flox/floxmice
tracedintothebasalforebrainand,inmiceinjectedwithAAV-GFP,fociofVglut2double-labeling(yellow)wereseeninaxon
terminals.AftertheAAV-GFP-ires-CreinjectioninaVglut2flox/floxmouse,thegreenterminalsnolongercontainedevidenceof TodeletetheVglut2genefocally,wein-
Vglut2staining,althoughmanyotherVglut2(cid:4)terminals(red)areseeninthebackground.Scalebars:A,B,20(cid:1)m;insetsinA,B, jected 42 homozygous Vglut2flox/flox
10(cid:1)m;C,50(cid:1)m;D,12.5(cid:1)m;E,F,10(cid:1)m. micestereotaxicallywithanAAVcontain-
ing the gene for Cre recombinase, either
Thenextday,thesectionswererinsedin2(cid:6)standardsalinecitrate aloneorincombinationwithGFP.Ascon-
(SSC)with50%formamidefor30minand,afterrinsingwithRNase trols,wemadesimilarinjectionsinfivewild-typelittermatesand
buffertwice,werethentreatedwithasolutionofRNaseA(20(cid:1)g/ml)at injectedeightVglut2flox/floxmicewithAAV-GFP(withoutCre).As
37°C for 30 min. After rinsing in RNase buffer twice and repeated
inourearlierworkwithAAVinjections(Chamberlinetal.,1998),we
washinginthe2(cid:6)SSCwith50%formamide,thesectionswerethen
sawnoevidenceoflocalinflammationattheinjectionsites.
washedinTris-HCl-bufferedsaline,andthenincubatedovernightin
Asexpected,AAV-GFPinjections(ourcontrolfornonspecific
alkalinephosphatase-conjugatedanti-digoxigeninantibody(1:200).
AfterrinsinginTris-bufferedsaline(TBS),sectionswerereactedwith vectoreffects)inVglut2flox/floxmicedidnotaffectVglut2expres-
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate, sion(Fig.3A).However,injectionsofAAV-CreinthePBinthe
7632•J.Neurosci.,May1,2013•33(18):7627–7640 Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia
Vglut2flox/floxmiceresultedinfocaldele-
tionofVglut2expressioninthetransduced
neurons(Fig.3B).Doublestainingshowed
that essentially all neurons with Cre-
immunoreactive nuclei failed to show
Vglut2 mRNA by in situ hybridization
(Fig.3C,D),indicatingthattheexpression
of Cre reflects loss of Vglut2 expression
(Tongetal.,2007).
In animals injected with AAV-GFP,
wefoundthatGFP-immunoreactivePB
neuronalterminalsinthebasalforebrain
coexpressed Vglut2 immunoreactivity.
However, in mice injected with AAV-
GFP-ires-Cre, there was no Vglut2 ex-
pression in the GFP-immunoreactive
terminalsinthebasalforebrain(Fig.3F).
Thus,Vglut2proteinexpressionwasalso
deleted from the terminals of Cre-
transfectedneurons.
The Cre injection sites could be di-
videdintotwogroups:thosethatheavily
involved the respiratory part of the PB,
and those in which few if any neurons
were transduced in the respiratory PB.
Amongthecasesinvolvingtherespiratory
PB,wefound17thatincludedatleast60%
oftheneuronsinthePBel/lcsubnucleibi- Figure4. SchematicrepresentationoftheextentofneuronsexpressingCreinthePBcomplexofVglut2flox/floxmice.L1–L3,
laterally(Fig.2G).Withinthatgroupof17 R1–R3,ExamplesfromeachofthefivegroupsofmicecharacterizedinFigure2areshowninasinglecolumn,withthefirstthree
cases, we identified six centered on the sectionsineachcolumnrepresentingrostrallytocaudallytheleftsideofthebrain(L1–L3),andthenextthreetherightside(R1–R3).
PBel/lc(theVLPBgroup;Fig.2B,VLPB), ColumnsA–EcorrespondtothefivegroupsofinjectionsitesidentifiedinFig.2.TheoutlinesoftheCre-expressingregionsareshown
projectedagainstNissl-stainedtemplatesectionsandthedifferentinjectionsitesarenumbered.Scalebar,100(cid:1)m.
five cases including at least 50% of the
neuronsintheKFnucleusaswellasvari-
ablenumbersofMPBneurons(theVLPB(cid:4)KFgroup;Fig.2C),
andsixinwhichtheinjectionalsoincludedneuronsinthemedial recurrentcyclesofelevatedCO causedbriefarousalsinalmost
2
PB subdivision bordering the PBel, but not the KF subnucleus alltrialsincontrolmice.Arousalswerejudgedbycorticalactiva-
(theVLPB(cid:4)MPBgroup;Fig.2D).Variablenumbersofneurons tion(lossofdeltaactivityinEEG),whichisusuallyaccompanied
inthedorsalandcentrallateralPB(PBdl/cl)wereincludedinall byEMGactivation(Fig.1).
ofthesecases(Fig.2B–D).
Normocapniccontrols
TheinvolvementofPBMandPBdl/clneuronsintherespira-
Tocomparethelatencytoarousalbyhypercapniatothelatency
toryPBcaseswascontrolledforbytwosetsofinjectionsthatdid
ofspontaneousarousals,weperformedtrialsinwhichthechan-
notincludetherespiratoryPB(showedCreexpressionin(cid:8)20%
nels in the gas mixer were switched every 300 s but both the
oftheneuronsinthePBel/lcorKF;Fig.2G).Onesetofcontrol
channelsdeliverednormocapnicair(O 21%;N ,79%).Alltrials
injections(theDLPBgroup,n(cid:3)4;Fig.2E,DLPB)includedthe 2, 2
werescoredinwhichtheanimalshadbeeninNREMsleepforat
dorsal, central, ventral, and internal lateral subnuclei, but few
least 30 s before onset of the gas exchange. In these trials, the
cellsinthePBel/lcorMPB.Theothersetofcontrolinjectionswas
average latency to arousal in controls (AAV-GFP and AAV-
centered in the MPB, but also included cells along the needle
CreWT) and experimental animals ranged from 102 to 110 s
trackinthePBcl/dl(theDLPB(cid:4)MPBgroup,n(cid:3)8;Fig.2F),but
(representing the expected remaining time in a typical NREM
again,fewcellsinthePBel/lcorKF.
sleepbout)andwasnotsignificantlydifferentamongthetreat-
Thefullsetofinjectionsanalyzedinthesegroupsisshownin ment groups. As a result, in only (cid:2)10–15% of trials did the
Figure4.Notethatinsixcasestheinjectionsiteseitherwerenot
animals arouse within 30 s of the switch, representing the fre-
symmetricortheymissedthetarget.Thesecaseswerenotana-
quencyofspontaneousarousalduringthetest(Fig.5).
lyzedfurther.
Arousalswithrepetitive10%CO (hypercapnia)for30s
2
EffectoffocaldeletionsofVglut2inthePBonRCAs Exposuretothehypercapnicstimuluswokeupthecontrolani-
RCAprotocol mals after a mean of 19 s in the AAV-GFP and of 15 s in the
Mice chronically instrumented with EEG and EMG electrodes AAV-CreWTcontrolgroups;thegroupsdidnotdifferstatisti-
wereplacedinaplethysmographchamberthathadacontinuous cally.In(cid:2)60%oftrials,thecontrolmicearousedwithin15sof
flow of normocapnic air (baseline 2 l/min of 21% O ; balance onsetofCO exposure,andin100%oftrialsby30s(Fig.6).
2 2
N ). Every 300 s, the gas source was switched to a normoxic- However,inallthreegroupsinwhichVglut2deletioninvolved
2
hypercapnic(10%CO )orhypoxic-hypercapnicmixture(10% thePBel(Fig.2,VLPB,red;VLPB(cid:4)KF,blue;VLPB(cid:4)MPB,ma-
2
O and 10% CO ) to simulate apnea for 30 s, after which the genta),hypercapnicarousalsweresignificantlydelayed(F (cid:3)
2 2 (6,33)
source was switched back to normocapnic air (Fig. 1). These 6.39,p(cid:8)0.001),withmeanlatencytoarousalinallthreegroups
Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia J.Neurosci.,May1,2013•33(18):7627–7640•7633
eningofanimalstoCO wasdiminished
2
atvirtuallyeverytimepoint.Asaresult,
the VLPB, VLPB(cid:4)KF, and VLPB(cid:4)MPB
groups also showed significant increases
infailurestoarouseto30sCO stimula-
2
tion(F (cid:3)17.6;p(cid:8)0.001;forallthree
(6,33)
groups compared with AAV-GFP and
AAV-CreWT), while the DLPB and
DLPB(cid:4)MPBgroupsarousedwithin30s
onalmostalltrials,similartothecontrol
animals (Fig. 6C). In (cid:2)40% of trials in
whichtheanimalswithPBel/lcdeletions
didnotawakenduringthe30sCO stim-
2
ulus,theanimalssubsequentlyawakened
atapacesimilartoanimalsthathadnever
beenexposedtoCO (Fig.5B).Withinthe
2
groups including PBel/lc neurons, there
were no significant differences between
theanimalsinwhichtheKFortheMPB
werealsoincludedandthegroupinwhich
theywerenot.
To distinguish whether the deficit in
arousaltoCO inthegroupswithPBel/lc
2
deletionswasduetoreducedrespiratory
sensationoreffort,weexaminedtherela-
tionshipoftheirminuteventilation(re-
spiratory rate times volume) with
arousal.Wefoundnosignificantcorre-
lation of the minute ventilation during
the30sofhypercapniawiththelatency
to arousal (Pearson correlation coeffi-
cient,r(cid:3)(cid:5)0.063;p(cid:3)0.75).Thus,itis
unlikelythatsensationfromrespiratory
movementduringthehypercapniacon-
tributedtothearousal.
Arousalswithrepetitivehypoxic
hypercapnia(CO ,10%;O ,10%)
2 2
for30s
Because obstructive apneas produce
varyingdegreesofhypoxiaaswellashy-
percapnia,wetestedarepetitivehypoxic-
hypercapnic stimulus to see whether the
role of the PB in hypercapnic arousal is
affectedbytheoxygenlevel.IntheAAV-
GFP and AAV-CreWT control groups,
hypoxichypercapniacausedEEGarousal
after 14 and 10 s respectively (Fig. 7).
However, the experimental groups with
Figure5. Responsesofmiceintrialswhenthetestgasmixtureisnormocapnicair(O2,21%;N2,79%).A,Meanarousallatency injections involving the PBel/lc (VLPB,
foreachgroupofanimalswithVglut2deletionsinthePBcomplex,calculatedfromthepointwhenthechannelswereswitched.B, VLPB(cid:4)KF, and VLPB(cid:4)MPB) showed
Timecourseofarousalsforeachgroup.C,Percentageoftrialsthatshowedarousallatenciesof(cid:1)15,30,and40s.Thenumbers significantly longer latencies (F (cid:3)
werequitesimilarforallgroups,indicatingthattheVglut2deletionsdidnotaffectthelikelihoodoftheanimalswakingup 13.67;p(cid:8)0.001)toarousalinresp(6o,3n0s)eto
spontaneouslyduringa100speriodofobservedsleep.Thisfigurerepresentsthenormocapnicresponsesoftheanimals,which
the hypoxic-hypercapnic stimulus (Fig.
werethentestedforarousalstohypercapniaandhypoxichypercapniaasshowninFigures6and7.
7A).IntheDLPBandDLPB(cid:4)MPBdele-
tion groups, which omitted the PBel/lc,
beingapproximatelythreetimeslongerthanthatinintactcontrol theresponsetohypoxichypercapniawas
animals.ArousalsintheDLPBandDLPB(cid:4)MPBgroups,which similar to the response of the control group, as observed with
largely excluded the PBel/lc neurons, were not different from hypercapnia.Themeanlatencytoarousalforthegroupsinclud-
controls(Fig.6A).Toexaminethedistributionofarousaltimes, ingthePBel/lcwasagain2–3timesthatofthegroupsthatdidnot
weperformedacrossallthegroupsasurvivalanalysistocompare includeit.
theprobabilityofremainingasleepatdifferenttimepointsafter The time course of EEG arousals was also analyzed across
onsetoftheCO stimulus(Fig.6B),demonstratingthattheawak- groups(Fig.7B)and,aswiththehypercapnicarousal,theper-
2
7634•J.Neurosci.,May1,2013•33(18):7627–7640 Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia
centageofanimalswithPBel/lcdeletions
thatwereawakenedwasdiminishedatev-
erytimepoint.Similartothehypercapnic
response,on(cid:2)35%oftrialsanimalsinthe
groupsthatincludedthePBel/lcdeletions
failedtoarouseduringthe30sstimulus
(F (cid:3)19.41;p(cid:8)0.001;Fig.7C),some-
(6,30)
thing that almost never occurred in ani-
mals in which the PBel/lc was intact.
Again as with the hypercapnic stimulus,
among the groups including deletion of
Vglut2inPBel/lcneurons,theinclusionof
theKFortheMPBmadenoapparentdif-
ferenceatanytimepoint.Thus,theaddi-
tion of hypoxia to hypercapnia has very
littleeffectonthetimetoarousalinour
model,orontheimportanceofthegluta-
matergicneuronsinthePBel/lcinarousal
tobothtypesofstimulus.
Doescoexpressionofother
neurotransmittersinglutamatergic
neuronsinthePBcompensateforloss
ofVglut2expression?
Deletion of Vglut2 in the PBel/lc dimin-
ishedbutdidnotabolisharousaltoCO
2
(Fig. 8). However, the PBel/lc neurons
also express other potential neurotrans-
mitters that may contribute to arousal,
such as calcitonin gene-related peptide
(CGRP;Yasuietal.,1991).Thereforewe
conductedasecondsetofexperimentsto
killtheglutamatergicneuronsselectively,
by using a line of mice in which Cre-
recombinasehadbeenknockedinatthe
Vglut2locus(n(cid:3)6).Togeneticallytarget
killingoftheVglut2neurons,wemicroin-
jected a conditional AAV that expresses
onlymCherry(aredfluorescentprotein)
incellswithoutCre,butwhichexpresses
thelethalAsubunitofdiphtheriatoxinin
cells with Cre (AAV-lox-mCherry-lox-
DTA-WPRE).Theextentofneuronalloss
in the PB (n (cid:3) 3) as demonstrated by
Neu-Nstaining(Fig.8A)wascomparable
totheextentoflossofVglut2expression
Figure6. Aseriesofgraphscomparingthearousallatenciesofthemiceinthedifferentexperimentalgroupsduringand
intheVLPB(dl/cl/el)groupinthedele-
afterexposuretoahypercapnicgasmixture(CO,10%;O,21%;N,69%).A,Meanarousallatencycalculatedfromthe
tion experiments. The arousal latency to 2 2 2
pointofincreaseinCO inthechamber.B,Timecourseofarousalsthatoccurredindifferentgroupsafteronsetof
hypercapnia in these mice was 51 s and 2
hypercapniaandarecalculatedasthepercentageoftrialsinwhichtheanimalsremainedasleepateachtimepoint.C,
wasnotsignificantlydifferentfromthatof Percentageoftrialsineachgroupthatshowedarousallatenciesof(cid:1)15,30,and40s.Notethatthearousalsweredelayed
miceinwhichVglut2alonehadbeende- andtrialswithfailuretoarouseweremorefrequentinallthreegroups(VLPB,VLPB(cid:4)MPB,andVLPB(cid:4)KF)inwhichthe
leted in the VLPB group (Fig. 8B). The deletionsincludedthePBel/lc,butthatlatencytoarousalinanimalswithdeletionsthatavoidedthisarea(DLPBand
percentageoftrialsthatshowedfailuresto DLPB(cid:4)MPB)wassimilartocontrolgroups.*p(cid:8)0.05;**p(cid:8)0.01,comparedwiththecontrolgroup(AAV-GFP).#p(cid:8)
arousewithin30spoststimuluswasalsocom- 0.05,comparedwiththeAAV-CreWTgroup.InB,thecolorsof*and#,correspondtorespectivegroups,andrepresents
parabletotheVLPBgroup.Micewitheither significancecomparedwiththecontrols.ErrorbarsshowSEM.
Vglut2deletionorwithnearlycompletelossof
glutamatergic neurons in the PBel/lc, also EffectoffocaldeletionofVglut2inthePBonwake–sleep
showedsimilarprobabilitiesofsleepatvarioustimepointsafter Because a recent report demonstrated hypersomnia after large
CO onset (Fig. 8C). Thus the full effect of the loss of gluta- lesionsofthePB(Fulleretal.,2011),wewereconcernedthatthe
2
matergic neurons in the VLPB on CO -induced arousal ap- diminishedarousaltoCO afterVglut2deletionsfromthePBel/
2 2
pearstobeaccountedforbythelossofglutamatergicsignaling lc might represent a generalized increase in sleepiness in these
fromthePBel/lc. animals. We therefore assessed spontaneous sleep and wake in
Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia J.Neurosci.,May1,2013•33(18):7627–7640•7635
our mice at 5 weeks after injection of
AAV-Cre (when the Vglut2 deletion
approached its maximum) and com-
paredthistowake–sleepincontrolan-
imalsaswellastheexperimentalgroups,
recorded 1 week after injection (when
preliminary time course experiments
showedminimalevidenceofVglut2de-
letion).Interestingly,thefocaldeletionof
Vglut2intheVLPBandDLPBgroups,
whichdidnotincludetheMPB,hadno
significanteffectonsleepamount,while
deletions that included the MPB
(DLPB(cid:4)MPB,VLPB(cid:4)MPB,andVLPB(cid:4)
KF) showed significant increases in
NREM sleep and reductions of wake
time(Fig.9A–C).
TheamountofNREMsleepdidnot
differbetweenmeasurementsinweeks
oneorfiveinthecontrolmice,includ-
ing both Vglut2flox/flox mice injected
withAAV-GFP,andwild-typemicein-
jectedwithAAV-Cre,withbothgroups
spendinganaverageof49.7%oftime
inNREMsleepduringthelightphase
and30%oftimeduringthedarkphase.
TheNREMsleeptimeintheVglut2flox/
flox mice injected with AAV-Cre also
didnotdifferfromcontrols1weekaf-
ter injection. By contrast, at 5 weeks,
significant changes in sleep amounts
emergedinthegroupsofVglut2flox/flox
animals injected with AAV-Cre into
theMPB.Duringthelightphase,only
theVLPB(cid:4)MPBgroupshowedamod-
erate increase in NREM sleep by
16.95% compared with week 1 AAV-
Crecontrol(F (cid:3)19.78,p(cid:8)0.001,
(6,72)
2-way ANOVA, followed by post hoc
testformultiplecomparisons).Dur-
ing the dark period, all three groups
that included deletions in the MPB
(DLPB(cid:4)MPB, VLPB(cid:4)MPB, and
VLPB(cid:4)KF) showed increases in
NREM sleep. The most pronounced
increase in NREM sleep was in the
DLPB(cid:4)MPBgroupwiththemostex-
tensive MPB Vglut2 deletion (42.8%
increase in NREM sleep compared
withweek1controlsand38.43%com-
Figure7. Aseriesofgraphscomparingthearousallatenciesduringandafterexposuretoahypoxic-hypercapnicgasmixture pared with week 5 controls; F (cid:3)
(6,72)
(CO,10%;O,10%;N,80%).A,Meanarousallatencycalculatedfromthepointofintroducingthehypoxic-hypercapnicgas 25.96, p (cid:8) 0.001, groups times 2-way
2 2 2
mixture.B,Timecourseofarousalsthatoccurredindifferentgroupsandarecalculatedasthepercentageoftrialsinwhichthe ANOVAfollowedbymultiplecompari-
animalsremainedasleepateachtimepoint.C,Percentageoftrialsineachgroupthatshowedarousallatenciesof(cid:1)15,30,and
sons). Comparatively smaller increases
40s.Notethatanimalsinallthreegroups(VLPB,VLPB(cid:4)MPB,andVLPB(cid:4)KF)withdeletionsincludingthePBel/lcshowed
in NREM sleep were seen in the
delayedarousalsandmoretrialswithfailuretoarouse,whereasthebehaviorofanimalswithdeletionsthatavoidedthisregion
(DLPBandDLPB(cid:4)MPB)wassimilartothatofcontrols.*p(cid:8)0.05and**p(cid:8)0.01,comparedwiththecontrolgroup(AAV- VLPB(cid:4)KF (36.18%) and VLPB(cid:4)MPB
GFP);#p(cid:8)0.05,comparedwiththeAAV-CreWTgroup.InB,thecolorsof*and#,correspondtorespectivegroups,and groups(34.20%),inwhichsmallernum-
representssignificancecomparedwiththecontrols.ErrorbarsshowSEM. bers of MPB neurons, mainly along its
lateralmarginnearthePBelandKF,were
affected.Overa24hperiod,theamount
of NREM sleep was also significantly
higher(F (cid:3)34.23;p(cid:8)0.001)inthe
(6,72)
7636•J.Neurosci.,May1,2013•33(18):7627–7640 Kauretal.•ParabrachialNucleusRegulatesArousalstoHypercapnia
DLPB(cid:4)MPB group (23.57%); followed
bytheVLPB(cid:4)MPBgroup(21.05%),and
the VLPB(cid:4)KF group (20.24%), com-
pared with 1 and 5 week controls (Fig.
9B). The Vglut2 deletion in the MPB
causedanincreaseinboththemeandu-
rationandnumberofNREMsleepbouts,
butbecauseofthevariancewithingroups,
neitherofthesereachedstatisticalsignifi-
cance.Thus,theincreaseinNREMsleep
amountprobablyreflectstheincreasesin
both bout duration and frequency, but
oursamplewasnotlargeenoughtomake
thisdistinction.
Reciprocal changes were seen in the
amountofthewakefulnessinthegroups
where the Vglut2 deletions included the
MPB (DLPB(cid:4)MPB, VLPB(cid:4)MPB, and
VLPB(cid:4)KF) (Fig. 9A). The amount of
REMsleep(Fig.9C)wasnotsignificantly
different in any of the groups compared
with the both AAV-GFP and AAV-Cre
controlsat1week.Inparticular,theani-
malswithdeletionsofthePBel/lc,butnot
including the MPB, showed the greatest
lossofCO -inducedarousal,buthadno
2
increaseinbaselinesleep.
EffectoffocaldeletionofVglut2inthe
PBondeltaactivity
To determine whether the mice with
Vglut2deletionsofthePBel/lcmighthave
haddeepersleepthatwouldhavecaused
them to be more difficult to awaken to
CO stimulation despite normal total
2
sleepamounts,wecomparedthepercent-
age of total EEG power in the delta fre-
quency range (0.5–4 Hz) across groups
Figure8. GeneticallytargetedkillingofLPBglutamatergicneuronsproducesreducedCO arousalsimilartodeletionofVglut2
whileawakeandduringNREMandREM 2
inthispopulation.A,PhotomicrographofabrainsectionimmunostainedforNeu-N,aneuronalmarker,afterbilateralinjectionof
sleep.Becauseitisdifficulttocompareab-
AAV-DTAkilledVglut2-CreneuronsintotheLPB.AlmostalloftheneuronsintheLPB,includingthePBel/lc,werelost(lesion
solutevaluesofEEGpoweracrossanimals
markedbydottedline).B,C,Graphscomparingthelatencyofarousalandprobabilityofsleepduringandafterahypercapnic
due to minor differences in electrode
stimulusof30sinmiceinjectedbilaterallywithAAV-DTA(green)comparedwiththecontrolmice(blackandgray)andtheVLPB
placement,eachanimal’sEEGpowerat1
groupfromwhichVglut2wasdeletedintheLPB,includingthePBel/lc(red),asshowninFigure6.NotethatthereductioninCO
week served as its own control and the arousalinthetwogroupswasessentiallyidentical.Scalebar,100(cid:1)m.*p(cid:8)0.05and**p(cid:8)0.01,comparedwiththecontro2l
percentage change from weeks 1 to 5 in group(AAV-GFP),and#p(cid:8)0.05,comparedwiththeAAV-CreWTgroup.InC,thecolorsof*and#correspondtotherespective
deltapowerwasanalyzedforeachanimal.
groupsandrepresentsignificancecomparedwiththecontrols.ErrorbarsshowSEM.
Thischangewasthencomparedacrossthe
groups. NREM EEG delta power signifi-
acousticstimulus.Atonewaspresentedatdifferentintensities
cantlyincreasedonlyinthetwogroupsthatincludedthelargest
(2,5,10,and30dB)for10severy300sinascendingorderof
partoftheMPB(VLPB(cid:4)MPBgroupandDLPB(cid:4)MPBgroup) intensity (Fig. 11). Although the VLPB(cid:4)MPB mice had re-
(F (cid:3)22.62,treatmentgroupstimestime,p(cid:8)0.001;Fig.9D).
(6,70) duced responses to CO and were sleepy (i.e., showed an in-
AtrendtowardanincreaseinthedeltapowerintheVLPB(cid:4)KF 2
crease in NREM sleep and delta power during NREM sleep
groupwasnotstatisticallysignificant.
during the day), their latency to arousal in response to the
DeltapowerduringwakefulnessandREMsleepwasnotsig-
soundwassimilartothatofthecontrolgroup.Atlowlevelsof
nificantlydifferentacrossallthegroups.Arepresentativehypno-
gramfromamouseintheDLPB(cid:4)MPBgroup(Fig.10)illustrates sound intensity (2 and 5 dB), both groups showed similar
numbersoffailurestoarouse,suggestingthattheyhadsimilar
theincreaseinEEGdeltapoweratweek5comparedwithweek1.
soundthresholds(i.e.,thePBdeletionshadnotaffectedhear-
Arousalsinresponsetoanacousticstimulus ing or the sound threshold needed to cause arousal). These
TodeterminewhethermicewithlargePBdeletionsweresim- experiments indicate that the delays and frequent failures of
ply hypoarousable in general, we examined the responses in arousaltohypercapniaorhypoxichypercapniaintheanimals
thegroupofmicewiththegreatestdeficitsinwakefulnessand with PBel/lc deletions were due to a selective insensitivity to
CO response (VLPB(cid:4)MPB group) and control mice to an respiratorychemosensorystimuli.
2