The Median Preoptic Nucleus Reciprocally Modulates Activity of Arousal-Related and Sleep ...

15 Pages·2007·1.12 MB·English

1616•TheJournalofNeuroscience,February14,2007•27(7):1616–1630
Behavioral/Systems/Cognitive
The Median Preoptic Nucleus Reciprocally Modulates
Activity of Arousal-Related and Sleep-Related Neurons in the
Perifornical Lateral Hypothalamus
NataliaSuntsova,1,2,4RubenGuzman-Marin,1,2SunilKumar,1,3Md.NoorAlam,1,2RonaldSzymusiak,1,3and
DennisMcGinty1,2
1ResearchService,VeteransAffairsGreaterLosAngelesHealthcareSystem,NorthHills,California91343,Departmentsof2Psychologyand3Medicine,
UniversityofCaliforniaLosAngeles,LosAngeles,California90095,and4A.B.KoganResearchInstituteforNeurocybernetics,RostovStateUniversity,
Rostov-on-Don344091,Russia
Theperifornical–lateralhypothalamicarea(PF/LH)containsneuronalgroupsplayinganimportantroleincontrolofwakingandsleep.
Amongthebrainregionsthatregulatebehavioralstates,oneofthestrongestsourcesofprojectionstothePF/LHisthemedianpreoptic
nucleus(MnPN)containingasleep-activeneuronalpopulation.ToevaluatetheroleofMnPNafferentsinthecontrolofPF/LHneuronal
activity,westudiedtheresponsesofPF/LHcellstoelectricalstimulationorlocalchemicalmanipulationoftheMnPNinfreelymoving
rats.Single-pulseelectricalstimulationevokedresponsesin79%ofrecordedPF/LHneurons.Nocellswereactivatedantidromically.
Direct and indirect transynaptic effects depended on sleep–wake discharge pattern of PF/LH cells. The majority of arousal-related
neurons,thatis,cellsdischargingatmaximalratesduringactivewaking(AW)orduringAWandrapideyemovement(REM)sleep,
exhibitedexclusivelyorinitiallyinhibitoryresponsestostimulation.Sleep-relatedneurons,thecellswithelevateddischargeduring
non-REMandREMsleeporselectivelyactiveinREMsleep,exhibitedexclusivelyorinitiallyexcitatoryresponses.ActivationoftheMnPN
viamicrodialyticapplicationofL-glutamateorbicucullineresultedinreduceddischargeofarousal-relatedandinexcitationofsleep-
relatedPF/LHneurons.DeactivationoftheMnPNwithmuscimolcausedoppositeeffects.TheresultsindicatethattheMnPNcontains
subset(s)ofneurons,whichexertinhibitorycontroloverarousal-relatedandexcitatorycontroloversleep-relatedPF/LHneurons.We
hypothesizethatMnPNsleep-activeneuronalgrouphasbothinhibitoryandexcitatoryoutputsthatparticipateintheinhibitorycontrol
ofarousal-promotingPF/LHmechanisms.
Keywords:perifornicallateralhypothalamus;medianpreopticnucleus;electricalstimulation;microdialysis;neuronalactivity;sleep–
wakingcycle
Introduction (QW)andnon-rapideyemovement(NREM)sleep(Estabrooke
Theposteriorlateralhypothalamuscontainsseveralinteracting etal.,2001;Torteroloetal.,2001;Alametal.,2002;Koyamaetal.,
neuronalpopulationscriticallyimplicatedinthecontrolofwak- 2003;Leeetal.,2005;Mileykovskiyetal.,2005).Currentevidence
ing,includinghistaminergic,glutamatergic,andpeptidergicneu- suggestsaroleofGABAergicinhibitioninthesleep-relatedinac-
rons(Lin,2000;GerashchenkoandShiromani,2004).Peptider- tivation of arousal-related PF/LH neurons and involvement of
gic cells producing hypocretins/orexins located within thismechanisminsleepcontrol.Itwasshownthatreversemicro-
perifornical–lateralhypothalamic(PF/LH)areaarethoughttobe dialysis of the GABAA receptor antagonist bicuculline into the
oneofthekeycomponentsofbrainarousalnetwork(deLecea PF/LHincreasedboththetimespentawakeandthenumberof
andSutcliffe,2005;JonesandMuhlethaler,2005;Sakurai,2005; c-Fos-positive hypocretin-immunoreactive and nonhypocretin
Saperetal.,2005). PF/LHcells(Alametal.,2005a).ApplicationoftheGABA re-
A
ThemajorityofPF/LHneurons,includinghypocretincells, ceptoragonistsintotheposterolateralhypothalamus(Linetal.,
exhibitincreasedneuronaldischargeandc-Fosimmunoreactiv- 1989;Sallanonetal.,1989)orthePF/LH(Thakkaretal.,2003)
ity during active waking (AW) compared with quiet waking causedhypersomnia.
RecentevidencesuggeststhatGABA-mediatedcontrolofthe
PF/LHcouldoriginateinsleep-activeneuronslocatedwithinthe
ReceivedMarch27,2006;revisedJan.8,2007;acceptedJan.9,2007.
preoptic area/basal forebrain. GABAergic cells showing sleep-
ThisworkwassupportedbytheMedicalResearchServiceoftheDepartmentofVeteransAffairs,NationalInsti-
tutesofHealthGrantsMH63323,MH47480,HL60296,andNS-50939,andtheJ.ChristianGillinResearchGrantfrom relatedc-Fosimmunoreactivityarefoundwithintheventrolat-
theSleepResearchSocietyFoundation(N.S.). eralpreopticarea(VLPO)(Sherinetal.,1996)andthemedian
CorrespondenceshouldbeaddressedtoDennisMcGinty,ResearchService(151A3),VeteransAffairsGreaterLos
preopticnucleus(MnPN)(Gongetal.,2004).Sleep-activeneu-
Angeles,HealthcareSystem,16111PlummerStreet,NorthHills,CA91343.E-mail:[email protected].
ronslocatedwithinthesenucleiexhibitaspecificpatternofen-
DOI:10.1523/JNEUROSCI.3498-06.2007
Copyright©2007SocietyforNeuroscience 0270-6474/07/271616-15$15.00/0 hanceddischargeduringbothNREMandrapideyemovement
Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity J.Neurosci.,February14,2007•27(7):1616–1630•1617
(REM)sleep(Szymusiaketal.,1998;Suntsovaetal.,2002),which thenacclimatedtoexperimentalmilieufor3d.Experimentswereper-
isoppositetothatdemonstratedbywake-activePF/LHneurons. formedonfreelymovinganimals.EEGandEMGsignalswererecorded
Warming of the preoptic area suppresses discharge of wake- bipolarly using Polygraph model 78 amplifiers (Grass Instruments,
activePF/LHneurons(Methipparaetal.,2003),whereasinhibi- Quincy,MA)withpassbandssetat1–30and100–1000Hz,respectively.
Neuronalactivitywasrecordedextracellularlyusingbipolarderivations
tionofthisareawithmuscimolinducesFosinhypocretinand
frommicrowires(impedanceat1kHz,500–800k(cid:2))andamplifiedbya
nonhypocretinPF/LHneurons(Satohetal.,2004).Recentana-
differentialACamplifier(model1700;A-MSystems,Carlsborg,WA)
tomicalstudiesshowedthattheMnPNinputtothePF/LHisone
withlowandhighcutofffiltersof10Hzand10kHz,respectively.During
oftheheaviestamongthestructuresinvolvedincontrolofbe- recordingsessions,themicrowireswereadvancedin25–30(cid:1)msteps,
havioralstates(Yoshidaetal.,2006).MnPN–PF/LHprojection untilactionpotentialswithsignal-to-noiseratio(cid:2)3wereobserved.
neurons include a subset of cells exhibiting sleep-related c-Fos Bioelectricalsignalsweredigitizedandstoredonharddriveforoff-line
immunoreactivity(Uschakovetal.,2006).Thesefindingsallow analysisusingMicro1401dataacquisitioninterfaceandSpike2software
ustohypothesizethatMnPNplaysanimportantroleinsleep- package(CambridgeElectronicDesign,London,UK).Polygraphicdata
relatedinhibitorymodulationofhypocretinandnonhypocretin weredigitizedatasamplingrate256Hzandunitactivitydataat10or25
arousal-relatedPF/LHneurons. kHzforwaveformandwavemarkdatachannels,respectively.
Electrical stimulation. Electrical stimulation of the MnPN was per-
An alternative or complementary source of inhibition of
formedwith200(cid:1)sconstant-currentsquarepulsesusingA-65Timer/
PF/LH arousal-related cells could be local GABAergic neurons
stimulatorcoupledwithSC-100constant-currentmonophasicstimulus
(Rosinetal.,2003)and/orcellscontaininganinhibitorypeptide
isolationunit(WinstonElectronicsCompany,Millbray,CA).Stimula-
melaninconcentratinghormone(MCH).Thelatterareintercon-
tionparameterswererestrictedbecauseMnPNstimulationatcurrent
nectedwithhypocretinneurons(Bayeretal.,2002)andarelikely intensities (cid:4)150–200 (cid:1)A triggered absence-like seizures within a few
to be a subset of PF/LH sleep-active cells (Verret et al., 2003; seconds.TotestPF/LHneuronalresponses,thefollowingnonepilepto-
Modirroustaetal.,2005). genicstimulationparadigmswerechosen.MnPNwasstimulatedat100
TodeterminethefunctionalinfluenceoftheMnPNonPF/LH (cid:1)A current intensity with single pulses (0.5 pulses/s) or 5 s trains of
neuronalpopulationswithdifferentrolesinbehavioralstatecon- stimulifollowedby150msperiod.Trainstimulationswereseparatedby
trol,weexaminedtheeffectsofelectricalstimulation,chemical atleast20srecoveryperiods.Stimulationfrequencywithinthetrainswas
stimulation,andchemicalinhibitionoftheMnPNonsingle-unit chosenbasedonmedianfrequencyofdischargeofMnPNsleep-related
neuronsinquietwakingdeterminedfromthepreviousdata(Suntsovaet
activitywithinthePF/LHinrats.
al.,2002).
Theeffectivecurrentspreadaroundthetipsofbipolarside-by-side
MaterialsandMethods electrodeshasnotbeenestimated.However,itislikelytobelessthanor
Animals.MaleSpragueDawleyrats(300–350g;8–10weeksofage)were atthemostcomparablewiththespreadfromatipofamonopolarelec-
housed individually in Stand-Alone Raturn Animal Handling System trode,whichwasestimatedasthesquarerootofthecurrentdividedby
(BioanalyticalSystems,WestLafayette,IN)placedinsideanelectrically thesquarerootoftheexcitabilityconstant(Tehovniketal.,2006).Ap-
shielded,sound-attenuatedchamber.Ratswerekeptina12hlight/dark plyingthisequation,monopolarstimulationwithparametersusedinour
cyclewithlightsonat8:00A.M.,designatedasZeitgebertime0(ZT0). studywouldbeexpectedtoactivatethemostexcitableneuronswithin0.6
Animalshadadlibitumaccesstofoodandwater.Allexperimentswere mmradius.Theeffectivecurrentspreadfromamonopolarelectrodehas
performedinaccordancewiththeNationalResearchCouncilGuidefor beenestimatedas250–500(cid:1)mbasedonsingle-cellrecordingsorusing
the Care and Use of Laboratory Animals. Animal use protocols were behavioralmethods(Wise,1972;BagshawandEvans,1976;Tehovniket
reviewedandapprovedbytheInternalAnimalCareandUseCommittee al.,2006).Comparedwithsomeofthesestudies,weusedlargerelectrode
oftheVeteransAffairsGreaterLosAngelesHealthcareSystem. tips,whichcouldresultinasmallereffectivecurrentspread(Follettand
Surgery.Surgicalprocedureswereperformedunderketamine/xylazine Mann,1986).Thisissupportedbyourfindingthat150–200(cid:1)mshiftsin
anesthesia(80/10mg/kg,i.p.respectively)andasepticconditions. electrodepositionfromtheMnPNmarginsinmediolateralordorsocau-
Forbehavioralstateassessment,stainless-steelscrewEEGelectrodes daldirectionsledtodisappearanceofEEG-synchronizingeffectofhigh-
wereplacedintotheskulloverthefrontalandparietalcortexandtwo frequency (100–200 pulses/s) MnPN stimulation and recruiting re-
Teflon-coatedstainless-steelEMGwireswereimplantedintothedorsal sponsesevokedbylow-frequencyMnPNstimulation.
neckmuscles. Themainadvantageofthemethodofelectricalstimulationisitshigh
To record a single-unit activity within the PF/LH, a preassembled temporalresolutionallowingthedistinctionofantidromic,monosynap-
constructionwasused.Itconsistedof10Formvar-insulatedstainless- ticandpolysynapticresponses.Alimitationislackofselectivityforneu-
steelmicrowires(20(cid:1)m)insertedintothe23-gaugeguidecannulathat rons. Possible excitation of axonal terminals and fibers of passage in
wasattachedtoamechanicalmicrodriveanchoredtotheminiatureelec- additiontoneuronalcellbodiesresultsinuncertaintyregardingtheori-
tricalconnector.Aholewastrephinedintheskull,centeredatanterio- ginofobtainedresponses.Toovercomethisproblem,wealsoapplied
posterior (AP), (cid:1)3.14; mediolateral (ML), 1.2 (Paxinos and Watson, localchemicalactivationandinhibitionofneuronalcellbodiesandden-
1998).Theguidecannulawasloweredinsidethebraintopositionitstip drites within the MnPN. This method, however, has lower temporal
3mmabovethetarget.Afterfixationoftheentireassemblytotheskull, precisionandpermitsassessmentofonlyrelativelylong-termtonicin-
the microwires were advanced through the guide cannula to a point fluencesofMnPNafferents.
correspondingtothePF/LHdorsalmargin[horizontal(H),8.2]. Reversemicrodialysis.MnPNcellularactivitywasmanipulatedchemi-
TostimulatetheMnPNelectrically,animalswereimplantedwithpar- callyusingmicrodialyticapplicationofL-glutamate,theGABAAreceptor
allelbipolarFormvar-insulatedtungstenelectrodes(80(cid:1)m;10–20k(cid:2)at antagonist bicuculline methiodide, and the GABA receptor agonist
A
1000Hz).Electrodewirestargetedthemostrostroventalanddorsocau- muscimol(allfromSigma,St.Louis,MO)toexcite,disinhibit,andin-
dalportionsoftheMnPN(AP,0.0;H,7.0;andAP,(cid:1)0.46;H,5,respec- hibittheMnPNneurons,respectively.
tively)(Swanson,1998)toprovidestimulationoftheentirestructure The microdialysis probe (cuprophane semipermeable membrane
(Fig.1A). length,1mm;outerdiameter,0.24mm;molecularweightcutoff,6000
To activate or inhibit the MnPN neuronal activity chemically, rats Da;CMA/11;CMAMicrodialysis,NorthChelmsford,MA)wasinserted
wereimplantedwithaguidecannulaforsubsequentinsertionofmicro- intotheimplantedguidecannula24hbeforestartinganexperiment.The
dialysisprobe.Theguidecannulawasimplantedinthemidlineata15° tipoftheprobeextended3mmbeyondthetipoftheguidecannula.The
anglefromtheverticalwiththetiplocated3mmfromthetarget(AP, positioningoftheprobewaschosentominimizedamagetothenucleus
(cid:3)0.12;ML,0;H,7). (Fig.1D).Thetipoftheprobe(coveredwithgluefor0.3–0.5mm)was
Recording.Theratswereallowedtorecoverfromsurgeryfor7d,and placedwithinthemostrostralpartoftheMnPN.Theopenpartofthe
1618•J.Neurosci.,February14,2007•27(7):1616–1630 Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity
microdialysismembranewasincontactwitha
dorsalsurfaceofthenucleus.
The probe was continuously perfused at a
flow rate of 2.0 (cid:1)l/min with filtered artificial
CSF(aCSF)containingthefollowing(inmM):
145NaCl,2.7KCl,1.3MgSO ,1.2CaCl ,and2
4 2
Na HPO ,pH7.2.Alldrugsweredissolvedin
2 4
this solution. Gastight Bee Stinger syringes
were filled with perfusates and mounted on
BabyBeeSyringePumpsconnectedtoBeeHive
Pumpcontrollers(BioanalyticalSystems,West
Lafayette,IN).Tochangesyringesduringmi-
crodialysiswithoutinterruptingflow,aliquid
switch(UniSwitch;BioanalyticalSystems)was
used.Afterdeliveryofsubstances,theperfusion
solution was switched back to aCSF and the
recordingcontinuedforanother45–90minor
untilthedischargeratewasatbaselinelevels.
To examine the effects of MnPN chemical
activationanddisinhibitiononPF/LHneuro-
nalactivity,1mML-glutamate,50(cid:1)Mbicucul-
line,and50(cid:1)Mmuscimolconcentrationshave
beenchoseninpreliminaryexperiments.These
concentrations,ononehand,werebelowthe
thresholdforinductionofEEGandbehavioral
manifestationsofabsence-likeseizuresbut,on
theotherhand,wereshowntobeeffectiveto
modulatebehavior,neuronaldischarge,c-Fos
immunoreactivity, and neurotransmitter re-
lease while applied by reverse microdialysis
into the other structures (Karreman and
Moghaddam, 1996; Materi and Semba, 2001;
Westetal.,2002;Satohetal.,2004;Alametal.,
2005a).ExcessesofeitherL-glutamateorbicu-
culline can potentially inactivate instead of
3
Figure 1. Anatomical localization of stimulating elec-
trodes,microdialysisprobes,andrecordedneurons.A,D,
Schematicdrawingshowingtargetedplacementofstimulat-
ingelectrodes(A)andmicrodialysisprobes(D)atsagittalsec-
tionoftheratbrain.B,E,Histologicallyverifiedlocationsof
thetipsofstimulatingelectrodes(B,asterisks)andmicrodi-
alysisprobes(E,verticalbars)plottedondiagramsofcoronal
sections.Thelocationsofthetipsofstimulatingelectrodes
withinrostralandcaudalMnPNinBareshownonthetopand
bottompanels,respectively.TheMnPNishighlightedwith
bluecolor.C,F,PhotomicrographsofNissl-stainedsections
showingthelocationsofstimulatingelectrodesandmicrodi-
alysisprobes.Thearrowsindicatethesitesofelectrolyticle-
sionswithintherostral(C,top)andcaudal(C,bottom)MnPN
andtrackofthetipofmicrodialysisprobewithintherostral
MnPN(F).Scalebar,250(cid:1)m.G,H,Locationsofneuronswith
differentsleep–wakedischargepatternsrecordedwithinthe
PF/LHduringexperimentswithelectricalstimulation(G)and
microdialyticperfusion(H)oftheMnPN.Forbettervisualiza-
tion,AW/REMsleep-relatedneurons(circles)areshownon
theleftsideandAW-related(triangles),NREM/REMsleep-
related (diamonds), REM sleep-related (rectangles), and
state-indifferent(asterisks)unitsareshownontherightside
ofthedrawings.Theperifornicalareaisoutlinedwithbrown.
I,Photomicrographsofstainedforhypocretin-1histological
sectionsshowingthelocationsofmicrowiretracks(arrows)
within lateral (1) and medial (2) parts hypocretin-
immunoreactiveneuronalfield.Scalebar,50(cid:1)m.MS,Medial
septalnucleus;ac,anteriorcommissure;och,opticchiasm;f,
fornix;VDB,nucleusoftheverticallimbofthediagonalband;
3V,thirdventricle;Br.,bregma.
Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity J.Neurosci.,February14,2007•27(7):1616–1630•1619
stimulateMnPNneuronsbecauseofdepolarizationblock.However,sig- thebeginningofdrugapplication.ThepercentagesoftimespentinAW,
nificantlyhigherconcentrationsofthesedrugshavebeenusedpreviously QW,NREM,andREMsleepwerecalculatedperhour.
tophysiologicallyactivatebrainregions(GeorgesandAston-Jones,2001; ToquantifytheeffectofchemicalmanipulationsoftheMnPNonthe
ZhangandFogel,2002;Hanamori,2003;Puigetal.,2003).Theconcen- dischargeofthePF/LHneurons,meanfiringrateswerecalculatedfor
trationofbicucullineusedinourstudy(0.025(cid:1)g/(cid:1)lintheperfusion sleep–wakestatesduringbaselineaCSFperfusionanddrugadministra-
fluidand(cid:5)10timeslessinthetissue)wasmuchlowerthanreportedto tion. Average firing rates obtained during different sleep–wake states
produceadepolarizationblockage(0.75(cid:1)g/(cid:1)l)inmicroinjectionexper- before and during drug application were compared with two-way
iments(Perieretal.,2002).Inaddition,inourpreliminarystudies,we repeated-measuresANOVAwithtwowithin-subjectsfactors.Onefactor
never observed inhibitory responses of preoptic area neurons to wasdefinedasasleep–wakestatewithfourlevels(AW,QW,NREM,and
L-glutamateandbicucullinelocallymicrodialyzedatconcentrationsused REMsleep).Theotherfactorwasdrugtreatmentwithtwolevels(aCSF
in this study. These preliminary experiments and our previous data
perfusionanddrugadministration).Incasesofviolationofsphericity
(Alametal.,2005a)basedonc-Fosanalysisallowedustoestimatethe
assumption (Mauchley sphericity test), to avoid erroneous results of
effective spread of drugs delivered by reverse microdialysis (0.5–0.75
univariateANOVA,amultivariateapproachwasappliedtotestthesig-
mmaroundtheprobe).
nificanceofunivariaterepeated-measuresfactorswithmorethantwo
Dataanalysis.Todeterminethestate-relatedchangesindischargeof
levels(Wilk’smultivariatetest).Inpresenceofasignificantmaineffect,
therecordedcells,themeanfiringrateswerecalculatedfor1015–60s
posthoccomparisonsweredoneusingTukey’sHSDtest.
artifact-freeperiodsofAW,QW,NREM,andREMsleep,whichwere Resultsareexpressedthroughoutasmean(cid:7)SEM.Thelevelofsignif-
identifiedonthebasisofEEGandEMGparametersusingstandardcri-
icancewassetatp(cid:6)0.05.
teria(Timo-Iariaetal.,1970).Recordedcellswereclassifiedintogroups
Histology.Attheendofarecordingsession,underdeepanesthesia
withdifferentsleep–wakefiringrateprofilesbasedontheresultsofa
(pentobarbital;100mg/kg,i.p.),microlesionsweremadeatthetipof
K-meansclusteranalysisfollowedbyevaluationofstate-relatednessfor
microwires(20(cid:1)Aanodaldirectcurrent;15–20s)atthemostventral
eachmemberofthecluster(Suntsovaetal.,2002).One-wayANOVA
recordedsite.Afterinjectionwithheparin(500U,i.p.),animalswere
followedbyTukey’shonestlysignificantdifference(HSD)posthoctest
wasusedtoexaminetheinterstatedifferencesinthemeanfiringratesof perfusedtranscardiallywith30–50mlof0.1Mphosphatebuffer,pH7.2,
followedby300mlof4%paraformaldehydeinphosphatebuffercon-
individual neurons and groups of cells with different sleep–wake dis-
chargeprofiles. taining15%saturatedpicricacidsolution,100mlof10%sucrose,and
TheorthodromicresponsesofPF/LHneuronsevokedbyMnPNstim- finally100mlof30%sucroseinphosphatebuffer.Thebrainswerere-
ulationwerecharacterizedbymeasuringtheonsetlatencyandduration movedandequilibratedin30%sucrose.Serialcoronalsections(40(cid:1)m)
of stimulation-induced effects from peristimulus time histograms werestainedforNissl(cresylviolet)toverifythepositionofthestimu-
(PSTHs).TogeneratePSTHs,atleast80trialsofsingle-pulsestimulation latingelectrodes.Toverifythepositionofmicrowiretrackswithinthe
performedduringAWwereselected.Thetrialsweredividedinto1ms hypocretinneuronalfield,sectionsthroughthePF/LHwereimmuno-
bins,andactionpotentialsoccurringwithineachbinweresummedover stainedforhypocretin-1asdescribedpreviously(Alametal.,2002).
all stimulation trials. Significant excitatory or inhibitory poststimulus Reconstructions of tracts of stimulation electrodes, microdialysis
neuronalresponseswereidentifiedinthosebinswhosecountswere(cid:4)2 probes,andmicrowiresweremadewiththeaidofNeurolucidaimaging
SDsaboveorbelow,respectively,thebaselinemean(averagedovera system(MicroBrightField,Colchester,VT)guidedbytheratbrainatlases
prestimulus period of 200 ms). For slowly firing cells, inhibition was ofPaxinosandWatson(1998)andSwanson(1998)forPF/LHareaand
definedbya(cid:4)50%decreaseinthefiringratewithrespecttothepre- theMnPN,respectively.Accordingtoreconstructions,stimulatingelec-
stimulus value. The neuronal response onset latency and offset were trodes were correctly positioned in five rats (Fig. 1B,C). From these
definedasthefirstoffiveconsecutivebinsmeetingtheresponsecriterion animals,87cells,recordedwithinhypocretin-immunoreactiveneuronal
andnolongermeetingthecriterion,respectively.Fivemillisecondwin- fieldwithsignal-to-noiseratio(cid:2)3(Fig.1G),wereexaminedfortheir
dowsallowedustoidentifybothshort-andlong-durationresponses. sleep–wake discharge patterns and responses to the MnPN electrical
GiventhatconductiontimefromtheMnPNtothePF/LHisunknown, stimulation. The vast majority (85%; n (cid:8) 74) of these neurons were
neuronal responses with onset latencies less than (cid:5)15 ms potentially recordedlateraltothefornixandwereconsideredtobeintheLH.The
couldbeconsideredasantidromicormonosynaptic,takingintoaccount restofthecellswererecordeddorsalormedialtothefornix,withinthe
possibilityoflowconductionvelocity(StockerandToney,2005)and
perifornical and dorsomedial hypothalamic nucleus. Localizations of
slowsynaptictransmission.Short-latencyexcitatoryresponseswereop-
bothmicrodialysisprobes(Fig.1E,F)andmicrowires(Fig.1H)within
erationallydefinedasmonosynapticiflatencyjitter(SDofthetimefrom
thetargetswerehistologicallyconfirmedinthreerats.Responsesof54
thebeginningofthestimulusartifacttothebeginningofthefirstevoked
PF/LHneuronswithidentifiedsleep–wakedischargeprofilestochemical
spike)was(cid:6)0.5ms.Antidromiccriteriaincludedtheabilitytofollow
stimulationand/orinhibitionoftheMnPNwereanalyzed.
single pulses with a constant latency ((cid:6)0.1 ms latency jitter) and to
followhigh-frequencystimulustrainsat100–200pulses/s.
ToestimatetheeffectsofMnPNtrainstimulationonthefiringrateof Results
PF/LHneurons,PSTHs(0.1sbinwidth;250bins;summationofatleast EEGresponsestoMnPNelectricalstimulation
30stimulationtrials)andrasterdisplaysweregenerated,whichprovided
TheeffectsoftheMnPNsingle-pulseandtrainstimulationon
graphicrepresentationofneuronalactivityoccurringfrom5sbeforeto
PF/LHneuronalactivitywerestudiedduringactivewaking.Five
15saftertheendofthetrainstimulation.TogeneratePSTHs,pulses
secondtrainstimulationoftheMnPN(200(cid:1)s,100(cid:1)Asquare
initiatingtrainsofstimuliwereusedastriggers.Themeanfiringrates
werecalculatedfor5sepochsbeforeandduringstimulationaswellasfor pulseswith150msperiod)evokedrhythmicactivityresembling
threeconsecutiveepochsthatfollowedthestimulation.Thesemeasure- recruitingresponsesorspindle-likeactivityincombinationwith
mentsweredoneforeachindividualtrialandforaveragedtrials.The high-amplitude(cid:3)waves.Trainsofhigh-frequencystimuli(100–
statisticalsignificanceofchangesinthefiringrateforeachindividual 200pulses/s)alsotriggeredEEGsynchronizationwithinthefre-
neuron and for subsets of the cells was determined using one-way quencyrangeofsleepspindles.Singlestimuluspulsesofthesame
repeated-measuresANOVAwithtimeasarepeatedmeasurefollowedby
durationandintensityatfrequency0.5pulses/sfailedtoinduce
Tukey’sHSDposthoctest.
EEGsynchronizationinAW.However,inQWandNREMsleep,
To quantify the sleep–wake parameters during baseline conditions
stimulation with these parameters evoked slow (180–300 ms)
andchemicaltreatmentsoftheMnPN,EEGandEMGrecordingswere
scored on the basis of the predominant state within each 10 s epoch wavesfollowedbyrhythmicafterdischarges(seriesofwavesthat
during1hofpredrugaCSFperfusionandduring1hperiodstartingfrom occurredat10–25Hzandlastedupto1.5s).
1620•J.Neurosci.,February14,2007•27(7):1616–1630 Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity
Table1.SummaryofelectrophysiologicalresponsesofPF/LHneuronstoasingle-pulseMnPNstimulation
Initialresponse Secondresponse
Inhibition Excitation Excitation Inhibition
Number Latency Number Latency Duration Number Duration Number Duration
Sleep–wakedischargeprofile ofcells (ms) Duration(ms) ofcells (ms) (ms) ofcells (ms) ofcells (ms)
AW/REMsleep-relatedwithSPFM(n(cid:8)41) 31 6.5(cid:7)0.9 87.1(cid:7)7.7 10 44.2(cid:7)5.9
AW/REMsleep-relatedwithMFM(n(cid:8)16) 6 24.0(cid:7)1.1 119.3(cid:7)25.5 8 3.6(cid:7)0.4 5.3(cid:7)1.6 8 74.6(cid:7)14.4
AW-related(n(cid:8)5) 4 6.8(cid:7)2.5 82.3(cid:7)22.1
NREM/REMsleep-related(n(cid:8)9) 9 9.0(cid:7)1.8 9.1(cid:7)1.8 7 60.6.(cid:7)11.2
REMsleep-related(n(cid:8)9) 7 7.4(cid:7)1.2 11.4(cid:7)3.3 3 49.7(cid:7)8.1
State-indifferent(n(cid:8)7) 4 21.0(cid:7)1.9 30.5(cid:7)8.7
PF/LHneurons:sleep–wakedischargepatternsandresponses tionfollowedbyinhibition(Table1).Thepercentagesandrep-
toMnPNelectricalstimulation resentativeexamplesoftheseresponsesareshowninFigure2,C
The electrophysiological responses of 87 PF/LH neurons were andD–F,respectively.
analyzed. Exclusively inhibitory reactions or initial inhibitions were
InagreementwithpreviousPF/LHunitactivitystudies(Alam foundin37cells(82%ofresponsiveunits).Thevastmajorityof
etal.,2002;Koyamaetal.,2003),theexaminedcellswereheter- theseresponderswereSPFMneurons(n(cid:8)31).Theoverallmean
ogeneousintermsoftheirsleep–wakedischargeprofiles.Based responselatencyanddurationofexclusivelyandinitiallyinhibi-
ontheresultsofclusteranalysisfollowedbyevaluationofstate- toryresponsesareshowninTable1.
relatedness for each member of the cluster, PF/LH cells were Twenty-sevenAW/REMsleep-relatedcells(21SPFMneurons
subdivided into five groups designated in accordance with the and6MFMcells)exhibitedpureinhibitoryresponses(Figs.2D,
state(s) in which unit discharge was the highest, namely: 3B).K-meanclusteringanalysisshowedexistenceofthreeclus-
AW/REMsleep-related,AW-related,NREM/REMsleep-related, tersofunits(n (cid:8)10;n (cid:8)11;n (cid:8)6)differinginlatencyof
1 2 3
REMsleep-related,andstate-indifferentneurons. response(2.0(cid:7)0.2,11.5(cid:7)0.9,and24.0(cid:7)1.1ms,respectively).
Single-pulsestimulationoftheMnPNperformedduringAW ThefirsttwoclustersincludedSPFMneurons,whereasthelong-
eliciteddischargechangesin69ofthe87PF/LHcells(79.3%).No est latencies were found in MFM cells, which constituted the
cells were activated antidromically. Although the predominant thirdcluster(Fig.2G).Overallmeanonsetlatencyandduration
MnPN-inducedtransynapticeffectwasinhibitory,neuronswith ofpureinhibitoryresponseswere10.4(cid:7)1.7and105.1(cid:7)9.1ms,
differentsleep–wakedischargeprofilesexhibiteddifferentreac- respectively.
tionstostimulation.Characteristicsoftheseresponsesaresum- Biphasic inhibitory–excitatory responses (Fig. 2E) were
marizedinTable1. found only in SPFM cells (n (cid:8) 10; 18%). These neurons were
groupedintotwoclusters(n (cid:8)4;n (cid:8)6)withdifferentlatency
AW/REMsleep-relatedneurons ofinitialinhibitoryresponse1(1.5(cid:7)20.3and10.0(cid:7)0.7ms,re-
Sleep–wake discharge pattern. Fifty-seven PF/LH cells (65.5%)
spectively)(Fig.2H).Theperiodofdischargereductionlastedfor
firedathigherratesduringAWandREMsleepcomparedwith 57.7(cid:7)8.6msandwasshorter(p(cid:6)0.01,Student’sttest)thanin
QW and NREM sleep. The interstate differences in the mean purelyinhibitedSPFMcells(101.1(cid:7)9.3).Themeandurationof
firing rate of these cells as a group and sleep–wake discharge postinhibitoryexcitationwas44.2(cid:7)5.9ms.
profiles of individual units are shown in Figure 2, A and B,
Biphasic excitatory–inhibitory responses were found exclu-
respectively. sivelyinMFMneurons(n(cid:8)8;14%)(Figs.2F,3D).Theywere
Themajority(n(cid:8)41;71.9%)ofAW/REMsleep-relatedcells
excitedwithalatencyrangingfrom2to5ms(Fig.2I).Themean
exhibited a single-pulse firing mode (SPFM) across all sleep–
latencyanddurationofbothexcitatoryandinhibitorycompo-
wake states (Fig. 3A). The rest of AW/REM sleep-related units
nentsoftheseresponsesareshowninTable1.
(n(cid:8)16;28.1%)generatedsinglepulsesduringwakingandREM
During5sMnPNtrainstimulation,thevastmajority(84.2%)
sleep and a combination of single pulses and high-frequency ofAW/REMsleep-relatedcellssignificantly(p(cid:6)0.05,Tukey’s
(400–900pulses/s)burstdischargesduringNREMsleep.These
HSD post hoc test) decreased their firing rates to 7.1–74.8% of
cellswereidentifiedwithinthePF/LHforthefirsttimeandwere
prestimulation firing. No neurons were facilitated. The overall
designatedasmixedfiringmode(MFM)neurons(Fig.3C).An
meanfiringrateofrespondingAW/REMsleep-relatedcellswas
abilityofPF/LHneuronstogenerateburstsduringNREMsleepis reducedby42.7%duringstimulation(p(cid:6)0.001,Tukey’sHSD
compatible with the findings that some LH neurons express a
posthoctest).Dischargeremaineddecreasedby31.8%for5safter
low-voltage-activatedcalciumcurrent(Fanetal.,2000)andre- theendofstimulustrains(p(cid:6)0.001)andby15.4%duringa
ceiveinhibitoryinputsfromthereticularthalamus(Baroneetal., subsequent 5 s epoch (p (cid:6) 0.05) (Fig. 2J). The group of cells
1994).ThepercentageofAW/REMsleep-relatedcellswashigher
inhibitedbytrainstimulationincludedboththeneuronsreactive
amongtheneuronsrecordedwithinLH(70.3%)thanamongthe
tosingle-pulsestimulationandunitsunresponsivetosinglestim-
neurons recorded within the medial part of hypocretin-
uli(Fig.2K,L).Inthelattercase,itislikelythattemporalsum-
immunoreactiveneuronalfield(38.5%).AllcellswithMFMwere
mation of postsynaptic potentials was needed to suppress dis-
foundwithintheLH.
chargeofthetargetneuron.
Responses to stimulation. Forty-five AW/REM sleep-related
cells(79%)respondedtoasingle-pulseMnPNstimulation.The AW-relatedneurons
responsesfellintothreecategories:(1)apureinhibition,(2)an Sleep–wakedischargepattern.FivePF/LHcells(5.8%)exhibited
initialinhibitionfollowedbyexcitation,and(3)aninitialexcita- maximumfiringratesduringAW.Theinterstatedifferencesin
Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity J.Neurosci.,February14,2007•27(7):1616–1630•1621
the mean firing rate of these cells as a
group and sleep–wake discharge profiles
ofindividualunitsareshowninFigure4,
AandB,respectively.AllAW-relatedneu-
ronsexhibitedasingle-pulsefiringmode
ineachstageofthesleep–wakecycle.
Four AW-related cells were recorded
dorsal or medial to the fornix (Fig. 1G).
Their percentage within this region was
30.8%,whichiscomparablewiththatpre-
viouslyreported(Alametal.,2002).Lat-
eraltothefornix,onlyoneof74recorded
neurons (1.4%) was AW-related. Hypo-
cretin cells exhibit an AW-related dis-
charge profile (Lee et al., 2005; Mileyk-
ovskiyetal.,2005).Thelowpercentageof
AW-relatedneuronswithinthelateraldi-
visionofhypocretinfieldimpliesthatlat-
eralhypocretincellsareeitherselectively
active during specific waking behaviors
underrepresented in our experimental
conditionsorhaveadifferentsleep–wake
dischargeprofile.
Responses to stimulation. Four AW-
related neurons responded to a single-
pulseelectricalstimulationoftheMnPN
withpureinhibition(Fig.4C,D,H).Two
cells responded with 2 and 3 ms latency,
andtheothertwocellswereinhibited10
and12msafterstimulusonset(Fig.4E).
The mean onset latency and duration of
responsesareshowninTable1.AllAW-
relatedunitswereinhibitedduringMnPN
train stimulation. The firing rates de-
creasedto26.4–64.0%ofbaselinefiring.
TheoverallmeanfiringrateofAW-related
units decreased by 58.3% (p (cid:6) 0.001,
Tukey’sHSDposthoctest)duringstimu-
lationandremainedreducedduringtwo
consecutive5sepochssubsequenttostim-
ulationperiodby51.0%(p(cid:6)0.001)and
30.1% (p (cid:6) 0.05) compared with pre-
stimulationfiring(Fig.4F).
Figure2. SummaryofdischargecharacteristicsofAW/REMsleep-relatedPF/LHneuronsandtheirresponsestoMnPNelectrical
stimulation.A,Groupmean(cid:7)SEMdischargerates(spikes/second)ofAW/REMsleep-relatedcellsacrosssleep–wakestates.The Sleep-relatedneurons
meanfiringrateinAWandREMsleepwassignificantlyhigherthaninQWandNREMsleep(one-wayrepeated-measuresANOVA Sleep–wake discharge patterns. Eighteen
followedbyTukey’sHSDtest).B,Meandischargeratesofindividualneurons.C,Percentagesofdifferenttypesofresponsesto
PF/LH neurons (20.7%) were sleep-
MnPNelectricalstimulationexhibitedbyneuronswithSPFMandMFM.Notethelowproportionofnonresponsive(NR)unitsand
related. These cells were identified as ei-
thehighpercentageofcellsexhibitingpureinhibitoryresponses(Inh)inbothSPFMandMFMsubsetsofneurons.TherestofSPFM
ther NREM/REM sleep- or REM sleep-
neuronsexhibitedinhibitory/excitatoryresponses(Inh/Exc),whereastheremainderofMFMneuronsrespondedwithinitial
related, depending on the sleep state(s)
excitationalwaysfollowedbyinhibition(Exc/Inh).D–F,Representativeexamplesofrasterplots(toppanel)andPSTHs(bottom
panel)correspondingtoanInh(D),Inh/Exc(E),andExc/Inh(F)responsesaftersingle-pulseelectricalstimulationoftheMnPN.For duringwhichtheyexhibitedelevateddis-
allPSTHshereandinsubsequentfigures,electricalpulses(200(cid:1)s;100(cid:1)A)wereappliedattime0(arrowheads),andbinwidth chargecomparedwithwaking.
was1ms.Thescaleonthey-axisisinspikes/second.Rasterdisplayshowstheoccurrenceofspikesforeachtrialaccumulatedinthe NREM/REM sleep-related neurons
PSTH.They-axisintherasterplotshowsthenumberoftrials.G–I,Histogramsshowingdistributionsofonsetlatenciesforpure (n(cid:8)9;10.3%)wereforthefirsttimeiden-
inhibitory(G),initiallyinhibitory(H),andinitiallyexcitatory(I)responses.Noteasubstantialproportionofshort-latencyre- tified within the PF/LH. They increased
sponses.J–L,ResponsesofAW/REMsleep-relatedunitstotrainstimulationoftheMnPN.J,Groupmean(cid:7)SEMfiringrateof48 their discharge during both NREM and
unitscalculatedforfiveconsecutive5sepochsstartingwiththeepochimmediatelyprecedingstimulationperiod.Duringstimu-
REMsleepcomparedwithwakingstates.
lation200(cid:1)s,100(cid:1)ApulseswereappliedtotheMnPNwith150msperiodfor5s(horizontalbarnexttox-axis).Notethatthe REM sleep-related neurons (n (cid:8) 9;
groupmeanfiringratesignificantlydecreasedduringstimulationandcontinuedtobereducedfor10saftercompletionof
10.3%)increasedfiringratesduringREM
stimulation.K,Inhibitionbytrainstimulationofaneuronunresponsivetosingle-pulsestimulation.PSTH(0.1sbinwidth,
sleep compared with waking states and
summationof30stimulationtrials)andrasterdisplayprovidegraphicrepresentationofactivityofthiscelloccurringfrom5s
beforethebeginningto10saftertheendofthetrainstimulation.L,Firingratehistogram,unitactivity,EMG,andEEGduringa NREMsleep.Activationofcellsoccurred
singlestimulationtrialmarkedbythearrowinK.*p(cid:6)0.05;**p(cid:6)0.01;***p(cid:6)0.001;ns,nonsignificant. 8–46sbeforeREMsleeponset.Theinter-
statedifferencesinthegroupmeanfiring
1622•J.Neurosci.,February14,2007•27(7):1616–1630 Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity
ratesofsleep-relatedcellsandsleep–wake
discharge profiles of individual units are
showninFigure5,AandB,respectively.
All sleep-related neurons exhibited a
tonicfiringmodeacrosssleep–wakestates.
The percentages of sleep-related cells
amongneuronsrecordedmedialandlat-
eraltothefornixwerecomparable.
Responses to stimulation. All NREM/
REMsleep-relatedcellandsevenofnine
REMsleep-relatedneuronswererespon-
sivetotheMnPNsingle-pulsestimulation
exhibitingeitherexclusivelyexcitatoryor
excitatory–inhibitoryresponses(Fig.5C).
Table 1 shows the overall mean latency
anddurationofexcitatoryresponsesand
themeandurationofpostexcitatoryinhi-
bitions calculated separately for NREM/
REMsleep-andREMsleep-relatedunits.
None of these parameters differs signifi-
cantly between subsets of sleep-related
cells.
Pure orthodromic excitations were
found in two NREM/REM sleep-related
neurons(Figs.5D,6B)andinfourREM
sleep-related cells with latencies ranging
from2to18ms.SevenNREM/REMsleep-
andthreeREMsleep-relatedneurons(Fig.
5E)wereinitiallyexcitedwith2–15msla-
tencies.Thedistributionoflatenciesofex-
clusivelyandinitiallyexcitatoryresponses
of NREM/REM sleep- and REM sleep-
relatedunitsisshowninFigure5F.
Fourof14sleep-relatedcellsrespond-
ingwith(cid:6)15msonsetlatencywereoper-
ationallydefinedasmonosynapticallyac-
tivated because they exhibited (cid:6)0.5 ms
latency jitter and ability to follow high-
frequency stimulation at 70–85 pulses/s
(see Fig. 6B). The rest of the cells were
classifiedaspolysynapticallyactivated.
4
withEEGdesynchronizationonarousalsfromNREMsleep
(a).B,Responseofthesameneurontoasingle-pulsestim-
2
ulationoftheMnPN.PSTH(bottompanel),rasterplot(middle
panel),andarawtrace(toppanel)ofasinglestimulationtrial
markedbyarrowaredisplayed.Theneuronrespondedwitha
short-latency(3ms)pureinhibition.C,ThedischargeofMFM
cellacrossthesleep–wakingcycle.c,ExpandedtracingfromC
markedbyhorizontalbar.1–4,Unitactivitytracingscorre-
spondingtoepochsmarkedbyverticalbarsinC:1,NREM
sleep;2,transitiontoREMsleep;3,REMsleep;4,AW.Note
thatMFMneuronsdramaticallyincreasedtheirfiringrates
18–42sbeforeREMsleeponset(c)primarilybecauseofan
increaseindensityofhigh-frequencyburstdischarges(c,
Figure3. RepresentativeexamplesoftheresponsesevokedinAW/REMsleep-relatedPF/LHneuronswithSPFMandMFMby compare1,2).D,Responseofthesamecelltoasingle-pulse
MnPNelectricalstimulation.A,ThedischargeofSPFMcellacrossthesleep–wakingcycle.Hereandinsubsequentfiguresshowing MnPN stimulation. The neuron responded with a short-
polygraphicrecordingsthatillustratethesleep–wakedischargepatternsofPF/LHneurons,thefiringratehistogram(Rate), latency(4ms)excitationfollowedbyinhibition.Thetoppanel
electromyogram(EMG),andelectroencephalogram(EEG)aredisplayed.Thescaleonthey-axisinthefiringratehistogramsisin ontheleftsideshowsrowtracesoftwosinglestimulation
spikes/second.Thesleep–wakestates(wakefulness,NREMsleep,transitionfromNREMtoREMsleep,REMsleep)areindicated trialsmarkedbyarrowsinrasterdisplay(middlepanel)and
undertheEEGtracingsusingthicksolid,dashed,dot-dash,anddottedlines,respectively.Extracellularlyrecordedunitactivity ontheright-handsidefragmentofthePSTHfromthebottom
(Unit)isshownonexpandedtracingfromthesectionsmarkedbyhorizontalbars(a,a).Notethatthecellbelongstothesubset panelshowingathigherresolutiontheinitialpartofthe
1 2
ofSPFMcells(n(cid:8)15)thatdramaticallyincreasedtheirdischarge8–23sbeforeREMsleeponset(a)andalmostsimultaneously response.
1
Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity J.Neurosci.,February14,2007•27(7):1616–1630•1623
Theresponsesofsleep-relatedneurons
to train stimulation of the MnPN were
tested on six NREM/REM sleep- and on
fiveREMsleep-relatedunits.Firingrates
ofoneNREM/REMsleep-andthreeREM
sleep-related neurons were significantly
affectedbytrainstimulations(p(cid:6)0.001,
one-wayrepeated-measuresANOVA).All
reactions were excitatory and strictly
timed to the stimulation period. During
stimulation, NREM/REM sleep-related
unitincreaseditsdischargeby137%and
REM sleep-related neurons by 30–46%
(mean,38.9(cid:7)4.7%)comparedwiththe
prestimulation firing rate (p (cid:6) 0.001,
Tukey’sHSDposthoctest).Thefiringrate
was at its maximum at the beginning of
stimulationanddecreasedtothebaseline
level after 2–3 s. Every cell responsive to
trainstimulationexhibitedexclusivelyex-
citatoryreactionstosingle-pulsestimula-
tion, whereas the two cells that did not
show significant responses during train
stimulationexhibitedbiphasicexcitatory/
inhibitoryresponsestosinglepulses.
State-indifferentneurons
Sleep–wake discharge pattern. Seven
PF/LHcells(8%)withasingle-pulsefiring
modedidnotshowsignificantchangesin
the mean firing rate across sleep–wake
states(F (cid:8)1.9;p(cid:4)0.05).Individual
(3,18)
cells exhibited (cid:6)25% between-state
differences.
Responses to stimulation. Four state-
indifferentneuronsrespondedtoasingle-
pulse stimulation of the MnPN. They
showed pure orthodromic excitations,
whichstarted17–26msafterMnPNstim-
ulation.Themeanlatencyanddurationof
theseresponsesareshowninTable1.
Train stimulation of the MnPN had
significant effects only on discharge of
those cells that showed excitatory re-
sponse to single-pulse stimulation. The
meanfiringratesofindividualneuronsin-
creased during stimulation by 61–105%
(mean,88.3(cid:7)9.5%)comparedwiththe
baseline.Ateachofthethreepoststimula-
tionintervals(5seach),thefiringratedid
notdiffersignificantlyfromtheprestimu-
lationvalue(p(cid:4)0.05,Tukey’sHSDpost
Figure4. SummaryofdischargecharacteristicsofAW-relatedPF/LHneuronsandtheirresponsestoMnPNelectricalstimula-
tion.A,Groupmean(cid:7)SEMdischargeratesofAW-relatedneuronsacrosssleep–wakestates.ThemeanfiringrateinAWwas hoctest).
highercomparedwithQWandbothphasesofsleep(one-wayrepeated-measuresANOVAfollowedbyTukey’sHSDtest).B,Mean ComparisonoftypesofPF/LHneuro-
dischargeratesofindividualneurons.C,Percentagesofdifferenttypesofresponsestosingle-pulseelectricalstimulationofthe nalresponsesandtheirratiosinfiverats
MnPN.NotethatallresponderswereinhibitedbyMnPNstimulation.Inh,Inhibitoryresponses;NR,nonresponsive.D,Rasterplot with different localization of stimulating
andPSTHcorrespondingtoaresponsethatappearedwith10msonsetlatency.E,Histogramofdistributionofonsetlatenciesof electrodetipswithintheMnPN(Fig.1B)
responses.Notethepresenceoflatenciescompatiblewithamonosynapticlinkage.F,Groupmean(cid:7)SEMfiringrateofAW- didnotrevealdifferencesinresponsesof
relatedunitscalculatedforfiveconsecutive5sepochsbefore,during,andaftertrainstimulationoftheMnPN.Notethatneurons
either arousal- or sleep-related units. In
wereinhibitedduringstimulationperiod(markedwithhorizontalbarnexttox-axis)andfor10sthereafter.G,Thedischargeof
cases when stimulating electrodes were
AW-relatedcellacrossthesleep–wakingcycle.g,g,ExpandedtracingsfromGmarkedbyhorizontalbars.Onthetoprightcorner
1 2 misplaced((cid:5)200and400(cid:1)mlaterallyor
oftheexpandedtracingssuperimposedareallactionpotentialsthatappearedduringcorrespondingfragmentsofrecordings.Note
150 (cid:1)m dorsally/200 (cid:1)m caudally from
thatneuronincreasesitsfiringratebeforearousalfromNREMsleep(g)andawakeningfromREMsleep(g).H,PSTHandrasterplot
1 2
showingshort-latency(2ms)inhibitoryresponseofthiscelltosingle-pulseMnPNstimulation.*p(cid:6)0.05;***p(cid:6)0.001. MnPN margins), we observed different
1624•J.Neurosci.,February14,2007•27(7):1616–1630 Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity
EEG- and PF/LH neuronal responses.
Low-frequency stimulation of these sites
failed to induce EEG-synchronizing re-
sponses,whereashigh-frequencystimula-
tion caused EEG desynchronization.
Among arousal-related units, we did not
find cells exhibiting inhibitory responses
with(cid:6)15mslatency.NREM/REMsleep-
relatedneuronswereeitherunresponsive
(twoofthreecells)orinhibitedbystimu-
lationofextra-MnPNsites.
ResponsesofPF/LHneuronsto
chemicalstimulationandinhibitionof
theMnPN
Activation of MnPN neuronal popula-
tionswithoutinfluenceonpassingfibers
wasachievedby1hmicrodialyticperfu-
sionofexcitatoryaminoacidL-glutamate
ortheGABA receptorantagonist,bicu-
A
culline,atconcentrationsof1mMand50
(cid:1)M, respectively. Experiments were con-
ductedduringthedarkphaseofthelight/
darkcycle(ZT13–ZT16),becauseduring
applicationofthedrugsinthelightphase,
sustained waking episodes were absent
Figure5. Summaryofdischargecharacteristicsofsleep-relatedPF/LHneuronsandtheirresponsestoMnPNelectricalstimu-
and,therefore,theeffectsofMnPNchem- lation.A,Groupmean(cid:7)SEMdischargeratesofNREM/REMsleep-relatedneurons(blackbars)andREMsleep-relatedneurons
ical stimulation on discharge of wake-
(hatchedbars).ThemeanfiringrateofNREM/REMsleep-relatedcellssignificantlyincreasedinQWversusAW,inNREMsleep
active PF/LH neurons could not be esti- versusQW,andinREMversusNREMsleep.ThemeandischargeofREMsleep-relatedneuronsdidnotdiffersignificantlybetween
mated. Table 2 shows the percentage of AW,QW,andNREMsleepandincreasedinREMsleepcomparedwitheveryotherstate.B,Individualmeandischargeratesof
time spent in different sleep–wake states NREM/REMsleep-relatedneurons(solidlines)andREMsleep-relatedneurons(dashedlines)acrosssleep–wakestates.C,Per-
1 h before and during perfusion of centagesofdifferenttypesofresponsestosingle-pulseelectricalstimulationoftheMnPN.Notethatallresponderswereexclu-
L-glutamateandbicucullineaveragedfor sivelyorinitiallyexcitedbyMnPNstimulation.D,E,RasterplotsandPSTHscorrespondingtoapureexcitatoryresponseof
allexperimentsconductedduringthedark NREM/REMsleep-relatedcell(D)andanexcitatory–inhibitoryresponseofREMsleep-relatedcell(E)tosingle-pulseMnPN
stimulation.F,HistogramsshowingdistributionofonsetlatenciesofresponsesofNREM/REMsleep-relatedcells(blackbars)and
phase.
REMsleep-relatedcells(hatchedbars).Notethepresenceofcellsrespondingwithshortlatencieswithinbothsubsetsofsleep-
ToassesseffectsofMnPNdeactivation
relatedcells.NR,Nonresponsive;Exc,excitatory;Exc/Inh,excitatory/inhibitoryresponses.*p(cid:6)0.05;***p(cid:6)0.001.
on PF/LH neuronal activity, the MnPN
was microdialytically perfused with the
ingthetreatments(n(cid:8)12)werepooledtogetherwiththecells
GABA receptoragonist,muscimol,duringthelightphase(ZT3–
A recordedthroughouttheentireexperiment.Forallneuronsde-
ZT6)ofthelight/darkcycle.Applicationofthisdrugat50(cid:1)M creasingdischargeinresponsetochemicaltreatments(n(cid:8)6),
concentrationfor15minresultedinaprolongedAWstate,which
theabsenceofdataonrecoveryofthebaselinedischargeisem-
appeared 1–3 min after the start of muscimol perfusion and
phasizedbecauseofuncertaintyregardingthecauseofchangesin
lastedfor35–84min(mean,61.3(cid:7)3.9).Duringthe1hperiod
theactivityoftheseneurons.
that started from the beginning of drug application compared
with1hpredrugaCSFperfusion,animalsspent91%ofthetime AW/REMsleep-relatedcells
inAW,NREMsleepwasdramaticallyreduced(p(cid:6)0.001,one- Effects of chemical stimulation and deactivation of the MnPN
wayrepeated-measuresANOVA)andREMsleepepisodeswere werestudiedon35AW/REMsleep-relatedPF/LHneurons.
almostabsent(Table2). Thechangesinthegroupmeanfiringratesof12and8AW/
Perfusion of the MnPN with 50 (cid:1)M muscimol was used to REM sleep-related units in response to administration of
evaluate responses of AW-, AW/REM sleep-related, and state- L-glutamateandbicuculline,respectively,areshowninFigure7,
indifferentunits.Toexamineresponsesofsleep-relatedneurons, AandB.Theneuronalresponsestothesetreatmentsweresimilar.
20(cid:1)Mmuscimolwasappliedfor1hduringthelightsonphaseof Two-wayrepeated-measuresANOVAinbothcasesrevealedsig-
thelight/darkcycle(ZT3–ZT6).Withthislowerconcentration, nificantmaineffectsofwithin-subjectsfactors(sleep–wakestate
sustainedperiodsofNREMandREMsleepwerepresentduring anddrugtreatment)ondischargeofAW/REMsleep-relatedcells
drugadministration(Table2). and significant interaction between the factors. During
The effects of chemical stimulation and inhibition of the L-glutamateandbicucullineadministration,themeanfiringrate
MnPNwerestudiedon36PF/LHcellsrecordedthroughoutall of AW/REM sleep-related cells decreased compared with aCSF
thestagesoftheexperiment(baselineaCSFperfusion,drugtreat- perfusion(F (cid:8)45.7,p(cid:6)0.001;andF (cid:8)24.5,p(cid:6)0.01,
(1,11) (1,7)
ment,andpostdrugaCSFperfusion).Amongthesecells,allre- respectively). The effects of both treatments depended on the
sponsive units recovered their baseline discharge during post- state of the sleep–waking cycle (L-glutamate: F(3,9) (cid:8) 5.1, p (cid:6)
drugaCSFperfusion.Inaddition,wearereportingresponsesof 0.05,Wilk’smultivariatetest;bicuculline:F (cid:8)4.0,p(cid:6)0.05).
(3,21)
18neurons,whichwererecordedduringbaselineanddrugtreat- Discharge suppression during L-glutamate and bicuculline ad-
mentsonly.Neuronsincreasingornotchangingdischargedur- ministrationwasstatisticallysignificantonlyinAW(p(cid:6)0.001
Suntsovaetal.•EffectsoftheMnPNonPF/LHNeuronalActivity J.Neurosci.,February14,2007•27(7):1616–1630•1625
L-glutamate on discharge of an AW-
relatedPF/LHunit.Duringdrugadminis-
tration,comparedwithpredrugaCSFper-
fusion,thecellexhibiteda98%reduction
initsfiringrateandbecamevirtuallysilent
in AW, whereas during QW and NREM
sleepthechangesofdischargeratedidnot
exceed25%.REMsleepdidnotoccurdur-
ing drug perfusion. L-Glutamate treat-
mentchangedthefiringrateofoneoftwo
cells, which were lost during postdrug
aCSF perfusion. This neuron decreased
dischargeonlyinAWto57%ofbaseline
firing.
Inresponsetobicucullineadministra-
tion,oneunitalmostceasedtodischarge
during the whole period of drug perfu-
sion, whereas the other cell showed 44%
decreaseinitsfiringrateexclusivelydur-
ingAW.
During MnPN perfusion with musci-
mol, one neuron increased discharge by
43%,andtheotherwasunresponsive.
Sleep-relatedcells
Atotalof11sleep-relatedPF/LHneurons
weretestedforresponsestoMnPNperfu-
sionwithL-glutamate(n(cid:8)2),bicuculline
Figure6. RepresentativeexampleoftheresponseofanNREM/REMsleep-relatedPF/LHneurontoMnPNelectricalstimulation. (n(cid:8)3),andmuscimol(n(cid:8)6).
A,Thedischargeofthecellacrossthesleep–wakingcycle.a,ExpandedtracingsfromAmarkedbyhorizontalbar.Notethat
Microdialytic application of L-
NREM/REMsleep-relatedneuronsceasedtodischargeordramaticallyreducedtheirfiringratesafewsecondsbeforearousal.B,
glutamateresultedinadramaticincrease
ResponseofthiscelltoMnPNelectricalstimulation.Rasterplot,PSTH,superpositionofrawtracesoffiveconsecutivestimulation
inthefiringrateofaNREM/REMsleep-
trials(1),andneuronalresponsesto80(2)and100pulses/s(3)repetitivestimuliaredisplayed.Noteshortlatency(2ms),lowjitter
ofresponse(1),andabilityofthecelltofollowrepetitivestimuliat80pulses/s(2).At100pulses/s,theneuronrespondsonlyto relatedneuronto250,500,260,and190%
everyotherstimuli(3).Thesefindingssuggestmonosynapticactivation. ofbaselinefiringinAW,QW,NREM,and
REM sleep, respectively (Fig. 9A). An
REMsleep-relatedcellincreaseddischarge
and p (cid:6) 0.01, respectively; Tukey’s HSD test). Individually, in by28and42%inNREMandREMsleep,respectively.
AW in response to L-glutamate and bicuculline treatments, 92 Duringbicucullineperfusion,twoNREM/REMsleep-related
and85%ofneurons,respectively,exhibitedchangesintheirac- unitsincreasedtheirfiringratesby44and54%inAWandby44
tivitymeetingtheresponsecriterion((cid:4)25%changeinthefiring and120%inQW.DuringNREMsleep,thechangesintheactivity
rate).Nocellswereactivated.DuringL-glutamateandbicucul- ofonecelldidnotmeettheresponsecriterion;theotherneuron
lineperfusion,responsivecellsreducedtheirdischargeto34.7(cid:7) increasedfiringby63%.DuringREMsleep,spikeactivityofthese
4.0%(range,11–59%)andto58.5(cid:7)4.3%(range,44–73%)of cellsincreasedby25and29%.AnREMsleep-relatedneuronwas
baseline firing, respectively. The effect of L-glutamate on dis- unresponsivetobicucullinetreatment.
chargeofanindividualcellacrosssleep–wakestatesisshownin In response to muscimol perfusion, the discharge of one of
Figure8A.Allcellsrecordedduringbaselineanddrugtreatment two NREM/REM sleep-related cells decreased by 43 and 25%
only(n(cid:8)3)reducedtheirdischargeinresponsetoL-glutamate duringNREMandREMsleep,respectively,andby(cid:6)25%during
(n(cid:8)2)andbicuculline(n(cid:8)1)treatmentsto29–45%ofbaseline waking(Fig.9B).Aunitthatwasnotrecordedduringpostdrug
firingandwerenotincludedintostatisticalanalysis. aCSFperfusionalsoexhibitedareductionoffiringto27,34,and
TheeffectsofMnPNperfusionwithmuscimolwerestudied 45%ofbaselinevaluesduringQW,NREM,andREMsleep,re-
on 12 AW/REM sleep-related cells. During the muscimol- spectively.TwooffourREMsleep-relatedcells(onewithverified
induced AW state, the mean firing rate of these neurons as a recoveryofthebaselinedischarge,theothernotrecordedduring
group increased (F (cid:8) 23.9, p (cid:6) 0.001, one-way repeated- postdrug aCSF perfusion) exhibited 37 and 28% decreases in
(1,11)
measuresANOVA)(Fig.7C).Individually,75%ofcellsincreased firingratesexclusivelyduringREMsleep.TheremainingREM
theirdischargeonaverageto220.8(cid:7)48.5%(range,134–600%) sleep-related neurons did not exhibit criterion responses to
of baseline firing; the rest of the cells were unresponsive. The muscimol.
effect of muscimol perfusion on discharge of AW/REM sleep-
State-indifferentneurons
relatedneuronisshowninFigure8B.
Intotal,responsesofeightstate-indifferentPF/LHunitstochem-
AW-relatedcells icaltreatmentsoftheMnPNwereanalyzed.
ResponsesofsevenAW-relatedPF/LHneuronstomicrodialytic ThefiringrateoffivePF/LHstate-indifferentneuronswasnot
application of L-glutamate (n (cid:8) 3), bicuculline (n (cid:8) 2), and affectedbyMnPNperfusionwithL-glutamate(n(cid:8)2)andmus-
muscimol(n(cid:8)2)intotheMnPNwerestudied. cimol(n(cid:8)3)by(cid:4)10%.Onecellincreaseddischargeby28%in
Figure 8C shows the effect of MnPN perfusion with responsetomicrodialyticapplicationofL-glutamate.MnPNper-