Isoflurane affects brain functional connectivity in rats 1 month after exposure

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2021

Isoflurane affects brain functional

connectivity in rats 1 month after exposure

Stenroos, Petteri

Elsevier BV

Tieteelliset aikakauslehtiartikkelit

© 2021 The Authors

CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/

http://dx.doi.org/10.1016/j.neuroimage.2021.117987

https://erepo.uef.fi/handle/123456789/26834

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ContentslistsavailableatScienceDirect

NeuroImage

journalhomepage:www.elsevier.com/locate/neuroimage

Isoﬂurane aﬀects brain functional connectivity in rats 1 month after exposure

Petteri Stenroos

^a

, Tiina Pirttimäki

^a

, Jaakko Paasonen

^a

, Ekaterina Paasonen

^a

, Raimo A Salo

^a

, Hennariikka Koivisto

^a

, Teemu Natunen

^b

, Petra Mäkinen

^b

, Teemu Kuulasmaa

^b

,

Mikko Hiltunen

^b

, Heikki Tanila

^a

, Olli Gröhn

^a^,^∗

aA.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FI,-70211 Kuopio, Finland

bInstitute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI,-70211 Kuopio, Finland

a r t i c le i n f o

Keywords:

Brain plasticity Local ﬁeld potential Functional connectivity

Functional magnetic resonance imaging Isoﬂurane

Rats

a b s t r a ct

Isoflurane,themostcommonlyusedpreclinicalanesthetic,inducesbrainplasticityandlong-termcellularand molecularchangesleadingtobehavioraland/orcognitiveconsequences.Thesechangesaremostlikelyassoci- atedwithnetwork-levelchangesinbrainfunction.Toelucidatethemechanismsunderlyinglong-termeffectsof isoflurane,weinvestigatedtheinfluenceofasingleisofluraneexposureonfunctionalconnectivity,brainelectri- calactivity,andgeneexpression.

MaleWistarrats(n=22)wereexposedto1.8%isoﬂuranefor3h.Controlrats(n=22)spent3hinthesameroom withoutexposuretoanesthesia.After1month,functionalconnectivitywasevaluatedwithresting-statefunctional magneticresonanceimaging(fMRI;n=6+6)andlocalﬁeldpotentialmeasurements(n=6+6)inanesthetized animals.Awholegenomeexpressionanalysis(n=10+10)wasalsoconductedwithmRNA-sequencingfrom corticalandhippocampaltissuesamples.

Isofluranetreatmentstrengthenedthalamo-corticalandhippocampal-corticalfunctionalconnectivity.Cortical low-frequencyfMRIpowerwasalsosignificantlyincreasedinresponsetotheisofluranetreatment.Thelocalfield potentialresultsindicatingstrengthenedhippocampal-corticalalphaandbetacoherencewereingoodagreement withthefMRIfindings.Furthermore,alteredexpressionwasfoundin20corticalgenes,severalofwhichare involvedinneuronalsignaltransmission,butnogeneexpressionchangeswerenotedinthehippocampus.

Isoﬂuraneinducedprolongedchangesinthalamo-corticalandhippocampal-corticalfunctionandexpressionof genescontributingtosignaltransmissioninthecortex.Furtherstudiesarerequiredtoinvestigatewhetherthese changesareassociatedwiththepostoperativebehavioralandcognitivesymptomscommonlyobservedinpatients andanimals.

1. Introduction

Inpreclinicalandclinicalwork, generalanesthetics areroutinely usedduringsurgicalprocedurestopreventmovement,andtominimize experiencedstressandpain.Ideally,anestheticsworkbycausingtem- poraryandreversiblelossofconsciousnessandreactivitywithoutad- ditionalsideeﬀects.Generalanesthetics,however,altertypicalawake brainactivitybyactingonmultipleneuralcircuits(Brownetal.,2011, 2010).Functionalmagnetic resonanceimaging(fMRI)(Biswalet al., 1995) studiesreveal that anesthetics alternormal whole-brainfunc- tional connectivity in the resting state (Paasonen et al., 2018), the

Abbreviations.BOLD,bloodoxygenationleveldependent;BS,burstsuppression;CA1,cornuammonis1,DG,dentategyrus;EEG,electroencephalography;EPI, echoplanarimaging;ID,innerdiameter;LFP,localﬁeldpotential;SE,spinecho,S1L,primaryleftsomatosensorycortex,S1R,primaryrightsomatosensorycortex.

∗Correspondingauthor.

activityevoked byexternalstimulation (Paasonenet al., 2016),and the hemodynamic response (Martin et al., 2006), which is the ba- sis ofthefMRIsignal (Ogawaetal., 1990).Furthermore, theeﬀects of anesthetics canlast beyondtheduration oftheanesthesia. Inthe adultanimalandhumanbrain,anestheticsinduceirreversiblemolecu- lar,cellular,andgeneticchanges,leadingtolong-termstructuraland functional changes(Colonet al., 2017).These changes occur at the levelofionchannels,synapses,andcells,whichacttogethertomod- ulate neuronalnetworks, andmay lead tobehavioral and cognitive manifestations.Moreover,anestheticsplayapotentialroleinthegen- eration of postoperative cognitivedysfunction (Miller et al., 2018),

https://doi.org/10.1016/j.neuroimage.2021.117987

Received2July2020;Receivedinrevisedform16February2021;Accepted16March2021 Availableonline21March2021

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although there is currently no general consensus (Rasmussen et al., 2003).

Isoflurane,oneofthemostwidelyusedanestheticsinpreclinicalex- periments,hasbothacuteandchroniceffectsonbrainfunction.Acutely, itsuppresseswholebrainneuronalactivityandmodulatesfunctional connectivity (Liu et al., 2013a). Additionally, at high doses, isoflu- ranecausessynchronousburstsuppression(BS)activityin thecortex (Liuetal., 2011).Atthecellularlevel, isofluranetriggers secondary signalingcascadesleadingtochanges inproteinandgeneregulation (Colonetal.,2017).Severalgeneandproteinexpressionstudieshavere- portedincreasedneuroinflammation,changedmicrogliareactivity,and apoptosis(Buntingetal.,2015;Culleyetal.,2006;Edmandsetal.,2013; Kodamaetal.,2011;Lowesetal.,2017;Zureketal.,2014),andhisto- logicstudieshavedemonstratedimpairedsynapticstructure,decreased neuronaldensity,andincreasedneuronaldifferentiation(Brineretal., 2010; Erassoetal., 2013; Platholiet al., 2014)in the hippocampus orthecortexhourstodaysafterisofluraneexposure.Moreover,gene expressionchangesimportantforneuralplasticitycanoccurinparal- lelwiththese changes evendaysorweeks aftertheinitial exposure (Culleyetal.,2006;Iietal.,2016;Joksovicetal.,2015;Rammesetal., 2009a; Uchimoto et al., 2014; Zhong et al., 2015).Also, isoflurane increasesblood-brainbarrierpermeability(Tétraultetal.,2008)thus leavingthebraintissueexposedtoperipheralinflammatoryresponses (SafavyniaandGoldstein,2019).Further,manypreclinicalanimalstud- ieshavedemonstratedconsequencesofisofluraneonmemoryandbe- havior(Culleyetal.,2004;LinandZuo,2011;Rammesetal.,2009b; Stratmann et al., 2010; Uchimoto et al., 2014; Zhang et al., 2014; Zhongetal.,2015)uptoseveralweeksafter2–4hanesthesiainduction.

Nostudiestodate,however,haveevaluatedchroniceﬀectsofisoﬂurane anesthesiaonwhole-brainfunctionalconnectivity.

Therefore,weinvestigatedtheisoflurane-inducedlong-termmodula- tionofbrainfunctionbyfMRI,localfieldpotential(LFP)measurements, andgeneexpressionmapping1monthaftertheinitialexposure.The resultsprovideinsightsintothemechanismsunderlyingtheisoflurane- inducedlong-termeffectsonthebrain,andcanthusbeappliedtoeval- uatethelong-terminfluenceofanesthesiaonpreclinicalinvivostudy designs.

2. Materialsandmethods 2.1. Animals

All animal procedureswere approvedby theAnimal Experiment BoardinFinland,andconductedinaccordancewiththeguidelinesset bytheEuropeanCommissionDirective2010/63/EU.Atotalof44adult (300–500gatthetimeoftheisofluranetreatment)maleratswereused (RccHanWistar,purchasedfromLaboratoryAnimalCenter,University ofEasternFinlandandEnvigoLaboratories,Netherlands).Theratswere dividedinto3separategroupsforfMRI(n=12),LFP(n=12),andgene expression(n=20)studies.Ineachgroup,ratswereequallydivided intotheisofluranetreatmentgrouporcontrolgroupwithnoisoflurane treatment.Animalsfrom2providerswereequallydistributedtoboth controlandtreatmentgroupsforthegeneexpressionstudy.Therats weregroup-housed inventilatedcagesandmaintainedona12/12h light-darkcycleataroomtemperatureof22±2°Cand50%−60%hu- midity.Foodandtapwaterwereavailableadlibitum.

2.2. Isoﬂuranetreatment

Ratsintheisofluranetreatmentgroupwereexposedto1.8%isoflu- raneinO₂/N₂(30%/70%)for3hwhilepositionedontopofaheat- ingmattomaintainbodytemperature.Theanesthesiaconcentration andtimewereselectedtomimicalong-lastingsurgicalprocedure.Also, Colonetal.(2017)reportedthatexposureto1.2%to3.5%isoflurane anesthesiafor2to4hinducesbehavioral changesweekstomonths later.Adultratsinallgroupsweretreatedwithisofluranewithin1to

3-daytimeperiod.Controlratsweremovedtothesamelocationasthe isoflurane-treatedratsandkeptfor3hintheirhomecages,butwere nottreatedwithisoflurane.Onemonthaftertheisofluraneexposure,the firstgroupofratswasimagedwithfMRI,thesecondgroupwasusedin LFPmeasurements,andthethirdgroupwasusedforgeneexpression analysis(Fig.1).

2.3. fMRIprotocol

BeforetheMRImeasurements,cannulae(BDIntramedic^TM PE-10, FranklinLakes,NJ,USA)wereinsertedintothefemoralveinandartery underisoﬂuraneanesthesia(Attanevet1000mg/g,PiramalHealthcare UKLimited,Northumberland,UK;5%inductionand1.5%−2%mainte- nanceinamixtureofN₂/O₂70%/30%)forinjectingamusclerelaxant (~0.15mlof0.3mg/mlbolusand~1mgkg⁻¹ h⁻¹i.v.infusion,pan- curoniumbromide,Pavulon,Actavis)andsamplingthearterialblood (150μl).Atracheostomywasperformedformechanicallyventilating therats(Inspira,HarvardApparatus)tomaintainnormalbloodgasval- ues(analyzedwithi-STATModel300,AbbottPointofCareInc.,Prince- ton,NJ,USA).Thedurationofthecannulationandtracheostomypro- cedureswas30±6minfortreatedratsand28±5minforcontrolrats (p>0.70,t-test).

MRIwasconductedina7TBrukerPharmascanmagnetrunbyPar- aVision5.1softwareandequippedwithaBrukerquadratureresonator volumecoil(ID=72mm)andaratbrainquadraturesurfacecoil.Lo- calshimming ofthebrainwas conductedwitha3Dfieldmap-based method.fMRIwasperformedusingasingle-shotspin-echoechoplanar imaging(SE-EPI)sequencewiththefollowingparameters:TR2000ms, TE45ms,matrixsize64×64,field-of-view2.5×2.5cm,11slicesof 1.5-mmthickness,andabandwidthof250kHz.AnatomicT₂-weighted imageswereobtainedusingtheTurboRAREsequencewiththefollow- ingparameters:TR4680ms,echospacing16.13ms,8echoes,effective echotime48.4ms,matrixsize512×512,field-of-view5.0×5.0cm², 30slicesof0.75-mmthickness,andbandwidthof46.9kHz.

Resting-statefMRIwasrununder3isofluraneconcentrationsin5 measurementperiods:1.3%,2.0%,2nd1.3%,3.0%,anddeadanimal (Fig.1).A1.3%concentrationwas selectedtorepresentamoderate andtypicalisofluraneconcentrationaccordingtothepreclinicalfMRI literature(Haenseletal.,2015;Jonckersetal.,2015).Concentrationsof 2.0%and3.0%wereselectedtorepresentadeepisofluraneconcentra- tioncausingeasilydistinguishedBSneuronalactivity(Liuetal.,2011).

Eachperiodlasted10min(300volumes)andtherewasa10-minpause betweeneachperiodtoavoidcarry-overeffectsofthepreviousisoflu- raneconcentration.Animalsweresacrificedimmediatelyafterthemea- surementsbyinjectingsaturatedKCl(~0.3ml,i.v.)under5%isoflurane anesthesia.

2.4. fMRIanalysis

fMRIdatawerefirstconvertedtoNIfTI(http://aedes.uef.fi),then slice-time-andmotion-corrected,co-registeredtoaselectedreference brain, andsmoothed (2 × 2 voxel full-width at half-maximum) us- ingSPM8(https://www.fil.ion.ucl.ac.uk/spm/software/spm8).Regions of interests (ROIs) were drawnon thereference brain according to an anatomic atlas (Paxinos and Watson, 2004). Twelve ROIs were selected forthewhole-brainanalysis, includingauditorycortex,hippocampus,hypothalamus,medialfrontalcortex,medialthalamus,motorcortex,nucleusaccumbens,retrosplenialcortex,somatosensorycortex,striatum,ventrolateralthalamus,andvisualcortex(Supplementary Figure1).

Resting-statefunctionalconnectivityanalysiswasconductedusing the12ROI-template.Afterband-passfilteringat0.01-0.15Hz,aPear- soncorrelation(r)analysiswasperformedduringthe10-minresting- stateperiodsateachisofluraneconcentrationtocalculatethecorrela- tioncoefficientsbetweentheROIs.Additionally,correlationswerecal- culatedwithglobalsignalregressiontostudytheimpactofastrongBS

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Fig.1. Experimenttimelineandtheisofluraneadministrationschemeusedinthelocalfieldpotential(LFP)andfunctionalmagneticresonanceimaging(fMRI) measurements.Measurementswereconductedwithmechanicallyventilatedratsunderisofluraneconcentrationsof1.3%,2.0%,2nd1.3%and3.0%,andpost- mortem.

signalproﬁleoncorrelationvalues(SupplementaryFigure2).Theglobal signalwasobtainedfromthebrainmask,andpartialcorrelationcoeﬃ- cientswerecalculatedbetweentheROIsusingthepartialcorr-function inMatlab(R2011a).Motionregressionwasnotnecessaryastheanimals wereparalyzedandmechanicallyventilated.Calculatedr-valueswere transformedtoz-scoresbyFisherz-transformationpriortocalculating groupaveragesandperformingstatisticalcomparisons.Subsequently, thez-scoreswereconvertedbacktor-values.

ROI-basedcorrelationmatriceswereconstructedfromeachgroup, andagroup-leveldiﬀerencematrixwascalculatedbysubtractingthe meancorrelationvaluesofthecontrolratsfromthoseoftheisoﬂurane- treatedrats(Fig.4AandB).Thestandarddeviationsofthecorrelation valuesfrombothgroupsareillustratedseparatelyinamatrixformat (SupplementaryFigure3).

Tovisualizeﬁndingsobservedinthecorrelationmatrices,voxel-wise correlationmapsweregeneratedfromthesmoothed imagesbyusing seedregions in thesomatosensorycortex, hippocampus,andventro- lateralthalamus(SupplementaryFigure4).Valueswerethresholdedat r>0.1.fMRIsignalpowerspectrumanalysiswasperformedfromROIs inthecortexandhippocampus(Fig.5).Beforecalculatingthepower, thefMRIsignalwasnormalizedusingapercentsignalchangetransfor- mation.Statisticaltestswereperformedfromthesumofvaluesinthe 0.01-0.15Hzfrequencyrange.

2.5. LFPprotocol

Epidural screw(BN 650, BossardHolding AG,Switzerland) elec- trodesandhippocampaldoubleelectrodesmadeofstainlesssteelwire (50-μmdiameter,Nr.15168,CaliforniaFineWireCo.,GroverBeach,CA, USA)witha600-μmtipseparationwereprepared,electrochemically cleaned,andtested forimpedance(490±44Ω,and273±104kΩ, respectively).

Theratwasplacedintoastereotaxicframe(DavidKopfInstruments, Tujunga,CA,USA).Thescalpwasremoved,andtheskullwascleaned withsterilesaline,and3%hydrogenperoxide.Screwswereimplanted bilaterallyovertheleft(S1L)andright(S1R)(AP:−1,ML:+/-3mm) primarysomatosensorycortex.Adoubletwireelectrodewasimplanted intotherighthippocampusatthelocationsofrightdentategyrus(DG) (AP:−3.8,ML:+1.6,DV:−4.3mm)andrightCA1(AP:−3.8,ML:+1.6, DV:−3.7mm).Ascrewelectrode,usedasareference,wasplacedon topofthecerebellum(AP:−12,ML:−2mm).Theendoftheinsulated silverwire(0.2mmbarewirediameter)wasexposedandplacedunder theskintoserveasagroundelectrode.Afterelectrodeimplantation,the femoralveinandarterywerecatheterizedasdescribedinSection2.3.

Finally,atracheostomywasperformedandtheratswereventilatedto maintainnormalbloodgasvaluesandtoensuresimilarmeasurement conditionsasinthefMRIexperiments.Thetotaldurationoftheelec- trodeimplantation,cannulation,andthetracheostomyprocedureswas

106±8minfortreatedratsand99±8minforcontrolrats(p>0.30, t-test).

LFPwasrecordedwithSciWorksdataacquisitionsystem(Datawave Technologies,Loveland,CO,USA)witha2049-Hzsamplingrate.Mea- surementswereconductedwiththeidenticalanesthesiaprotocolused forthefMRImeasurements:ratswererecordedfor10minateachisoflu- raneconcentrationwith10-minbreaksbetweenperiodsandfinallywith thedeadanimal.Theorderofrecordingfromcontrolandtreatedrats was randomized.Animalsweresacrificedbyinjecting KClunder5%

isoﬂuraneanesthesiaasdescribedabove.

2.6. LFPanalysis

DatawereanalyzedwithMatlabandSpike2,version8.First,the directcurrent componentsof the signalwereremoved, signals were band-stopfilteredat49–51Hzusinganotchfilter,andband-passfil- teredat1–90Hzusingasecond-orderButterworthfilter(Spike2).LFP coherence(Fig.6)andaspectrogram(SupplementaryFigure5)were obtainedfromeachisofluraneperiodusingthemscohereandspectro- gramfunctions,respectively,inMatlabwitha4-swindowsizeanda 2-swindowoverlap.Forthecoherenceanalyzes,thevaluesfromeach frequencyband(delta,1–4Hz;theta,4–8Hz;alpha,8–13Hz;andbeta 13–30Hz)weresummatedseparately,andthebandsweresubsequently comparedbetweengroups.

BS activity from each channel was analyzed with a Mat- lab script FindRipples (http://fmatoolbox.sourceforge.net/Contents/

FMAToolbox/Analyses/FindRipples.html)thatwasfurthermodifiedin- house.Burstsweredetectedasepochsexceedinganamplitudeof4^∗SD abovethebaselinethathadamaximaldurationof2000msandamini- malinter-burstintervalof10msduring1.3%isofluraneanesthesia,and aminimalinter-burstintervalof400msin2.0%and3.0%isoflurane anesthesia.

Detectedepochswereusedtocalculateburstoccurrencerate(1/s), duration of suppression periods (s), and amplitudeof burstperiods (Fig.3A–C).Burstoccurrencerateswerealsoinspectedmanuallyfrom thedataobtainedunder2.0%isofluraneanesthesiatovalidatetheac- curacyoftheautomaticdetection.Theoccurrenceratesdidnotdiffer significantly betweenthemanual andautomaticdetection (p= 0.18 and0.41inthecontrolandtreatedgroups,respectively,t-test).Aver- agedburstoccurrenceratesandsuppressiondurationswerecalculated foreachbrainarea(S1L,S1R,DG,andCA1).Tostudytheamplitude ofburstactivity,thesuppressionperiodswerefirstremovedfromthe signal.Then,therootmeansquare(rms)functionofMatlabwasusedto obtainrmsvaluesfortheremainingsignalforeachisofluraneconcen- trationseparately.

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2.7. mRNAsequencing

Aftertheinitial isofluranetreatmentanda waiting periodof 30 days,animalswereperfused,andbrainregionswereextractedforthe mRNA-sequencinganalysis.Forbrainsampleextraction,theratswere firstanesthetizedwith5%isoflurane.Aneedlewasinsertedintotheleft ventricletopuncturethevenacava.Theanimalswereperfusedwith ice-coldNaClataflowrateof30ml/minforatotalof4min.Immedi- atelyaftertheperfusion,theheadwasdetachedwithaguillotine,the brainwasextractedfromtheskull,andregionsfromthesomatosensory cortexandhippocampusweredissectedunilaterally.Thetimebetween anesthesiainductionanddeathwas15to20min.Brainsamples(n=40) werequicklyfrozeninliquidnitrogenandstoredat−80°C.Theorder ofobtainingtissuefromcontrolandtreatedratswasrandomized.Later, thetissuewashomogenizedincoldphosphate-bufferedsalineandTRI- zolReagent,andtotalRNAwasextractedwiththeDirect-zol^TMRNA Miniprep(ZymoResearch,Irvine,CA,USA).

TotalRNAwasstoredat−80°Cbeforebeinganalyzedbystranded mRNAsequencingattheFinnishFunctionalGenomicsCentre(Turku CentreforBiotechnology,UniversityofTurkuandÅboAkademiUni- versity).First,thequalityofthesampleswasveriﬁedbytheAdvanced AnalyticalFragmentAnalyzer(Agilent)andtheRNAconcentrationwas measuredwithQubit® FluorometricQuantitation(LifeTechnologies).

TheRNAlibrarywaspreparedusingaTruSeq® StrandedmRNASample PreparationKit(Illumina).The40librarieswithgoodquality(≥85%

basesaboveQ30)wererunin2lanes.Samplesweresequencedwith 260–320 M reads/lane (minimum of 13 M reads/sample)and with single-readlengthof1×50bp(IlluminaHiSeq3000).Rawdatawas deliveredinfastq-format.

2.8. Geneexpressionanalysis

First,sequencingadaptersequenceswereremoved,andthereads werequality-trimmed(Trimmomaticversion0.38)(Bolgeretal.,2014) and mapped against the ribosomal reference sequences (build 6.0) (Bowtie2version2.2.3)(LangmeadandSalzberg,2012).Successfully mappedreadswerethenabandoned.Theremainingreadswerealigned withtheratreferencetranscriptome(build6.0)andtranscriptabun- dancequantiﬁcation was performed(Kallisto v.0.44.0) (Bray et al., 2016).

Abundanceestimates werethencollapsedtogene-levelcounts (R packagetximportversion1.10.1,R3.5.1)(Sonesonetal.,2016)sepa- ratelyfromthesomatosensorycortex(n=20)andhippocampus(n=20) ofindividualanimals.Countswerepre-ﬁltered(atleast3sampleswitha countof5orhigher)andnormalized(RpackageDESeq2version1.22.2) (Loveetal.,2014).

Finally,geneontologyanalysiswasperformedbycomparingthesig- nificantlydifferentlyexpressinggenesandgeneclassestoothergenes andgeneclasses from studies investigatingshort-term (days) effects of isoflurane anesthesia (Bunting et al., 2015; Culley et al., 2006; Dingetal.,2017;Edmandsetal.,2013;Lowesetal.,2017;Zureketal., 2014).First,inthePantherclassificationsystem(PANTHER14.1),com- mon genesandclasses fromotherstudieswerefilteredby statistical overexpressiontestsbasedonbiologicprocessontology(p>0.05,FDR corrected,Fisher’sexacttest),Thestatisticallyoverexpressedgenesand classeswerethencomparedwithgenesandclassesfromourstudy.

2.9. Statisticalanalysis

Statisticaltestingforcorrelationmatrices,fMRIpower,LFPcoher- ence,theburstoccurrencerate,suppressionduration,andburstampli- tudewasconductedbyperforming2-tailed2-samplet-tests(p<0.05)in Matlab,usingafalsediscoveryrate(FDR)toaccountformultiplecomparisons.Thediﬀerentiallyexpressedgenes(log2fold-changes)between theisoﬂuranetreatmentandcontrolgroups(SupplementaryFigure6) wereanalyzedwithlikelihoodratiotestinDESeq2separatelyfromthe

somatosensorycortex(n=10+10)andthehippocampus(n=10+10).

TheFDRwasusedtoaccountformultiplecomparisons.Alldataareex- pressedasmean±SD.Thep-valuesareindicatedas^∗<0.05,^∗^∗<0.01, and^∗^∗^∗<0.001.OriginaldataofthisstudyareavailableatMendeley Data(http://dx.doi.org/10.17632/wxftdp4n6x.1).Allcodesareavail- ableondemandbyemailingthecorrespondingauthor.

3. Results

Noneoftheanimalswereexcludedfromtheanalysesduetomove- mentoranatomicalanomalies.

3.1. Physiologicparameters

Noneofthemeasuredphysiologicparametersdifferedsignificantly between theisoflurane-treatedand controlrats. Inthe fMRI experi- ments,thevalues wereasfollows: pCO₂ (39.4±3.9controlmmHg, 44.9±4.9mmHgtreated;p=0.401),pO₂(148.7±11.7mmHgcon- trol,145.8±12.1mmHgtreated;p=0.870),andpH(7.32±0.03con- trol,7.32±0.03;p=1.000).IntheLFPexperiments,thecorrespond- ingvalueswereasfollows:pCO₂ (41.5±3.2mmHg control,41.4± 3.4mmHgtreated;p=0.972),pO₂(155.2±18.7mmHgcontrol,157.7

±14.0mmHgtreated;p=0.917),andpH(7.42±0.02control,7.39± 0.02treated;p=0.326).

3.2. Burstsuppression

ElectrophysiologicalandfMRIdatawereobtainedunder3different isofluraneconcentrations(1.3%,2.0%,and3.0%).Representative2-min periodsoffilteredLFPsignalsfromananimalinthecontrolandanani- malinthetreatedgroupduringanesthesiawitheachisofluraneconcen- trationareprovidedinFig.2.Asexpected,isoflurane-relatedinter-burst intervalswereshortduringthelowestanestheticconcentrations(1.3%), becamelongerunder2.0%isoflurane,andonlyoccasionalburstswere visibleinacontinuouslysuppressedstateunder3.0%isoflurane.The typicalburstlengthwas1–2sandintra-burstfrequenciesrangedfrom 1to40Hz.

TheparametersrelatedtoBS,analyzedfromtheLFPdata,areshown in Fig.3.Theoccurrencerate,durationofBSperiods,andburstam- plitudes didnot diﬀersigniﬁcantlybetween groupsin eithertheso- matosensorycortex(1.3%:p>0.47;2.0%:p>0.94;3.0%:p>0.22, FDRadjusted)orhippocampus(1.3%:p>0.38;2.0%:p>0.86;3.0%:

p>0.13,FDRadjusted).

3.3. Functionalconnectivity

Functionalconnectivityresultswithoutglobalsignalregressionare summarizedingroup-levelmatrices(Fig.4).Asageneralobservation, functionalconnectivity differedbetween groupsin thehippocampal- cortical,thalamo-cortical,andcortico-striatalconnections.Differences wereclearestwiththe2.0%isoflurane,whilesomesignificantdiffer- enceswerealsoobservedwith1.3%isoflurane.Correspondingresults with global signal regression areshown in Supplementary Figure 2 wheresimilar,butslightlyweaker,findingscanbeobservedwhencom- paredtotheresultsobtainedwithoutglobalsignalregression.

Cortico-corticalfunctionalconnectivitydidnot differsignificantly betweengroups(p≥0.07;Fig.4).Isofluranetreatment,however,af- fected striatal connectivity with the visual cortex and hippocampus (p<0.05FDRadjusted).Interestingly,hippocampal-corticalconnectiv- itydisplayedwidegroup-leveldifferences(5of6ROIpairs,p<0.05 FDRadjusted).Similarly,theconnectivityoftheventrolateralandme- dialthalamuswithcorticalareasexhibitedwidegroup-leveldifferences (10of12ROIpairs,p<0.05FDRadjusted).

ThecorrelationmapsshowninSupplementaryFigure4show the ﬁndingsfromthecorrelationmatrices.Thecortico-corticalconnectivity patternappearedspatiallysimilarbetweenthegroups,buthippocampal

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Fig.2. RepresentativeLFPsignalsundereachisoﬂu- raneperiodobtainedfromprimarysomatosensorycor- texfromananimalinthecontrol(A)andananimalin thetreatedgroup(B).Eachpanelshow2minofﬁl- teredLFPsignal.

andthalamicconnectivitydemonstratedspatialdifferencesbetweenthe groups.AfterFDRcorrection,however,nosignificantvoxel-wisediffer- encesremained.

3.4. fMRIpower

ThefMRIpowerspectraareshowninFig.5.Thecorticalpowerin- creasedsignificantlyduringthe2.0%(p=0.01)at0.01–0.15Hz.No statisticallysignificantdifferencesweredetectedinthepowerspectra ofthehippocampusineitherisofluraneperiod(p≥0.28).

3.5. LFPcoherence

LFPcoherence(1–30Hz)betweentheDGandS1RorS1Lisshown inFig.6.Intheisofluranetreatmentgroup,thecoherencebetweenthe DGandS1Rwasincreasedinthealpha(8–12Hz;p=0.037,FDRad- justed)andbetabands (12–30Hz;p= 0.027,FDRadjusted)during thefirst1.3%isofluraneperiod.Increases werealsoseenin thebeta band(p=0.049,FDRadjusted)duringthe2.0%isofluraneperiod.LFP coherencebetween theDGandS1L wasincreasedinthealphaband (p=0.015,FDRadjusted)duringthefirst1.3%isofluraneperiod.There werenosignificantdifferencesinLFPcoherencebetweentheCA1and S1R/S1L(p>0.05).

3.6. Geneexpression

Genesexhibitingsignificantlydifferent group-levelexpressionare showninTable1.Inthesomatosensorycortex,1.8%isofluranetreat- mentinducedsignificant(adjustedp-value<0.05)differentialexpres- sionof20genes:7showeddownregulatedexpressionand13showedup- regulatedexpression.Fold-changesvariedbetween−0.58to1.62,and averagefold-changewas|0.50|±0.08.Notably,nosignificantchanges ingeneexpressionweredetectedinthehippocampus.

4. Discussion

Theeﬀectsofanesthesiacanlastdaysandweeksbeyondtheanesthe- siatermination,whichhasimplicationsforexperimentaldesigninlab- oratoryanimalworkandforpatientcareinclinicalsettings.Themain

novelfindingofthepresentstudywasthatasingleisofluraneexposure induceslong-termnetwork-levelchangesinbrainfunctionalconnectiv- itythatlastatleast1month.Substantialchangesweredetectedinthe thalamo-corticalandhippocampal-corticalconnectivityobservedwith both fMRIandLFPtechniques.Thegeneexpressionfindings suggest thatthesefunctionalchangescouldbepotentiallyrelatedtoalterations inspecificionchannelsandsynapsesregulatingneuronaltransmission inthecortex.Thefunctionalconnectivitychangeswerenotlikelycaused bydifferencesintheanesthesiadepthbecausenogroup-leveldifferences weredetectedintheisoflurane-inducedBSoccurrencerate,durationof suppression,orburstamplitudes.

4.1. Functionalconnectivityunderisoﬂuraneanesthesia

Isoﬂuranemodulatesresting-statefunctionalconnectivitycompared withtheawakestateinrats(Liuetal.,2013a;Stenroosetal.,2018).

Under moderate isofluraneanesthesia, brain excitatory neurons are suppressedwhile inhibitory neuronalactivityis enhanced(deSousa et al., 2000; Dong et al., 2006), which may explain the altered resting-state functionalconnectivity. Underisofluraneconcentrations of 1.25%−2.0%, brain activity can shift toa BS state (Hudetz and Imas, 2007; Liuet al., 2011; Vincentet al., 2007) with alternating high-voltage peaks(bursts)andsilentstates(suppression)with little tonoelectricalactivity(Derbyshireetal.,1936).Oneexplanationfor thehyperexcitabilityduringBSiscompleteremovalofcorticalinhibi- tionbycorticalinterneurons(Ferronetal.,2009),leadingtounregu- lated neuronalfiringin thecortex.However,thebrainmayactively processinformationevenduringtheBSstatebyattemptingtorecover normalneuronaldynamicsduringtheburstphase(Chingetal.,2012; Japaridzeetal.,2015).Corticalburstsmaybeoriginatingbyrhythmic thalamocorticaloscillations(Steriadeetal.,1994;Zhangetal.,2019) orfrominternalbrainactivitysuchashippocampalgamma-oscillations (KroegerandAmzica,2007).

A hypersynchronized BS pattern is typicallyobserved in fMRIas highlystrengthenedcortico-corticalfunctionalconnectivity(Liuetal., 2013a; Paasonen et al., 2018; Pavel et al., 2015) whereas cortico- subcorticalfunctionalconnectivityis typicallysuppressed(Liuetal., 2011; Pawela et al., 2008). Our ﬁndings from fMRI and LFP mea-

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Fig.3. Burstoccurrence rate(A), durationofburst suppression periods (B), and burst amplitudes(C). Burstandsuppressionepochsweredetectedwithan in-housemodiﬁed Matlab script FindRipples. Aver- agesforburstoccurrencerate(1/s),suppressionpe- riod(s)andamplitude(mV)werecalculatedforcorti- cal(leftandrightprimarysomatosensorycortex)and hippocampalregions(dentategyrusandCA1).Statisti- caltestingwasdonewithatwo-samplet-testwithFDR correctionformultiplecomparisons.n.s.=nostatisti- calsigniﬁcance.Valuesaregivenasthemean±SD.

surementsunderisoﬂuraneanesthesia demonstratesimilarresults of widespread and strong cortico-cortical andmoderate cortico-striatal functionalconnectivityinfMRIwhilethehippocampal-corticalconnec- tivitywassuppressedonlyintheLFPdata.

Interestingly,whenminimizingtheimpactofBSactivityonfMRIsig- nalswithglobalsignalregression,highcortico-corticalfunctionalcon- nectivityin1.3%and2.0%isofluraneanesthesiaandmoderatecortico- striatalfunctionalconnectivityin2.0%isofluraneanesthesiawerestill preserved.This observationis consistent withprevious findings that functionalconnectivityisdetectedinthecortexandbetweenthecortex andsubcorticalstructuresevenafterregressingtheBSpatternfromthe fMRIsignal(Zhangetal.,2019).

4.2. Increasedfunctionalconnectivityinresponsetoisoﬂuranetreatment

Functionalconnectivitychangesinthalamo-corticalnetworkswere detectedinresponsetoisoﬂuranetreatment.Earlyexposureto1.5%

isoﬂuraneisreportedtoprolongplasticitybyreducinginhibitoryand increasingexcitatorypostsynapticpotentialsinthereticularthalamic

nucleus,causingabnormalthalamo-corticaloscillations(Joksovicetal., 2015).Interestingly,inourstudy,acomparableconcentrationof1.8%

isofluranecausedprolongedchangesinfunctionalconnectivitybetween thethalamusandcortexthatweredetectableafter1month.Notably, 1monthafterisofluraneexposure,manyofthethalamo-corticalfunc- tionalconnectivitychangeswerestilldetectedwhenminimizingtheim- pactofBSactivitywithglobalsignalregression.Thissuggeststhatthe detectedfunctionalconnectivitychangesarenotonlyprominentunder aBSstate,butarealsopresentaspartoftheintrinsicbrainstatethat occursunderisofluraneanesthesia.

Althoughisoﬂuranegenerallydecreaseshippocampal-corticalfunctionalconnectivity(Liangetal.,2015;Paasonenetal.,2018),preserved hippocampal-corticalfunctionalconnectivityhasbeenreportedtooc- curundertheBSstate(Liuetal.,2013b).Inourstudy,hippocampal- corticalfunctionalconnectivitywasstronglysuppressedinfMRIbutstill detectablewithLFP.Inresponsetoisoﬂuranetreatment,hippocampal- corticalfunctionalconnectivitywasstrengthenedmoderatelyin1.3%

and2.0% anesthesiain both LFPandfMRI. This mayimply altered informationtransferbetweenthehippocampusandcortexinresponse

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Fig.4. Thegroup-levelfunctionalconnectivity(A)anddifference(B)matricesobtainedfromisofluranetreatedandcontrolanimalsundereachisofluraneperiod. Statisticaltestingwasdonewithtwo-samplet-testwhileaccountingformultiplecomparisons(FDR).^∗=FDR-adjustedp-values<0.05.AC,auditorycortex;HC, hippocampus;HTH,hypothalamus;mFC,medialfrontalcortex;ThM,medialthalamus;MC,motorcortex;Nacc,nucleusaccumbens;RSC,retrosplenialcortex;SC, somatosensorycortex;Str,striatum;ThVL,ventrolateralthalamus;VC,visualcortex.

Fig.5. fMRIspectralpowerincortex(A)andhippocampus(B)ineachisoﬂuraneperiod.(A)Averagepowerfromtheleftandrightprimarysomatosensorycortices areshown.(B)AveragehippocampalpowercalculatedfromdentategyrusandCA1.Statisticaltestingbetweenthegroupswasdonewithatwo-samplet-test(whole 0.01–0.15Hzfrequencyrange).^∗p=0.05.Valuesaregivenasthemean±SD.

toisoﬂuranetreatment.Multiplestudiesreportprolongedmemoryor learningimpairments severalweekstomonthsafter2–4 hisoﬂurane treatment(Culleyetal.,2004;LinandZuo,2011;Stratmannetal.,2009; Uchimotoetal.,2014;Zhangetal.,2014;Zhongetal.,2015),which couldindicatemodulatedhippocampal-corticalfunction.Interestingly, functionalconnectivitychangesinourstudyweredetectablewithina comparabletimewindowof 1month.Severalstudiesreportedapop-

tosisorneuroinﬂammationin thehippocampusinresponsetoisoﬂu- rane treatment(Caoetal., 2012;Ge etal.,2015; Kongetal.,2013; Zhangetal.,2015),andthereforetheexactmechanismforpotential behavioralconsequencesofincreasedhippocampal-corticalfunctional connectivityfoundinthisstudyremainsunclear.

Reduced neural inhibition by GABA antagonists acutely in- creaseslong-rangecorticalandcortico-striatalfunctionalconnectivity

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Fig.6.Localﬁeldpotentialcoherencebetweenrightdentategyrusandright(A)orleft(B)primarysomatosensorycortices.Valuesfromeachfrequencyband(delta, theta,alpha,beta)weresummatedseparately,andbandsweresubsequentlycomparedbetweenthetreatmentgroups.Statisticaltestingwasdonebytwo-sample t-testwhileaccountingformultiplecomparisons.Weusedfalsediscoveryrate(FDR)toaccountformultiplecomparisons.^∗=FDR-adjustedp-values<0.05.S1, primarysomatosensorycortex;DG,dentategyrus.Valuesaregivenasthemean±SD.

Table1

Isoﬂuranetreatment(1.8%)inducedanupregulation(positivefoldchange)ordownregulation(negativefoldchange)ofcorticalgenesthatwasobserved 1monthlater.

Ensembl gene ID RGD gene ID Description Unadjusted p-value Adjusted p-value LOG2 fold change ENSRNOG00000015393 Slc32a1 Solute carrier family 32 member 1 2.523E − 08 0.000 − 0.215

ENSRNOG00000020346 Best1 Bestrophin 1 4.044E − 07 0.003 1.104

ENSRNOG00000008337 Gjd2 Gap junction protein, delta 2 1.910E − 06 0.007 − 0.584

ENSRNOG00000058739 Snn Stannin 1.914E − 06 0.007 − 0.172

ENSRNOG00000029262 - – 4.230E − 06 0.013 − 0.004

ENSRNOG00000014303 Lrp11 LDL receptor related protein 11 8.569E − 06 0.016 − 0.107 ENSRNOG00000026604 Cercam Cerebral endothelial cell adhesion molecule 8.312E − 06 0.016 0.732 ENSRNOG00000031766 Mt-cyb Mitochondrially encoded cytochrome b 7.751E − 06 0.016 0.733 ENSRNOG00000019598 Vegfa Vascular endothelial growth factor A 1.152E − 05 0.017 0.255

ENSRNOG00000057498 - Long non-coding RNA 1.071E − 05 0.017 0.567

ENSRNOG00000006639 Scn9a Sodium voltage-gated channel alpha subunit 9 1.362E − 05 0.018 1.620 ENSRNOG00000009661 Tgds TDP-glucose 4,6-dehydratase 1.618E − 05 0.020 0.390 ENSRNOG00000009039 Trappc12 Traﬃcking protein particle complex 12 1.852E − 05 0.021 − 0.341 ENSRNOG00000009156 Tra2a Transformer 2 alpha homolog 2.131E − 05 0.023 0.441 ENSRNOG00000007034 Hipk2 Homeodomain interacting protein kinase 2 2.642E − 05 0.026 0.588

ENSRNOG00000025075 Relt RELT, TNF receptor 3.167E − 05 0.029 − 0.391

ENSRNOG00000005641 Fbxl4 F-box and leucine-rich repeat protein 4 3.507E − 05 0.031 − 0.229

ENSRNOG00000053070 - Long non-coding RNA 4.688E − 05 0.039 0.488

ENSRNOG00000020250 Pcgf6 Polycomb group ring ﬁnger 6 6.551E − 05 0.049 0.462 ENSRNOG00000032902 Ybx1-ps3 Y box protein 1 related, pseudogene 3 6.294E − 05 0.049 0.615

(Nasrallahetal.,2017).Intheirstudy,theauthorsfoundincreasedfunc- tionalconnectivitybasedonbothfMRIconnectivityandEEGcoherence, andincreasedBOLDandevokedpotentialsinresponsetoforepawstimu- lation.Consistently,inthepresentstudy,onecortico-striatalconnection showedincreasedfunctionalconnectivityinfMRIandthecortexshowed increasedBOLDpower,2featuresthatpointtoincreasedneuralexcita- tion.Furthermore,partofourgeneexpressionresults(discussedbelow) suggestreducedneuralinhibition.Thispartoftheresultremainsuncon- clusive,however,because,onthebasisofLFPpower(datanotshown) orburstamplitude,neuralexcitationwasnotincreased.

Inadditiontotheneuronalconsequencesofisofluraneanesthesia, isofluranechangesregionalbloodflow(Hansenetal.,1988),andin- ducesincreasedblood-brainbarrierpermeabilityinthethalamusand cortex(Tétraultetal.,2008),potentiallyleadingtovasogenicedema and/orinflammatoryresponses,whichinturncanaltertissueexcitabil- ity(SchoknechtandShalev,2012)thatmaybereflectedinfunctional connectivity1monthlater.Theresultsof ourgeneexpressionanaly- sis,however,didnotdirectlysupportaninflammatoryresponse-related cascade.

4.3. Modulatedsignaltransmissionsuggestedbygeneexpressionﬁndings

Inthepresentstudy,5ofthe20geneswithisoflurane-modulatedcor- ticalexpressionbelongtothesamegeneclassesidentifiedearlierasex- hibitingshort-termisoflurane-inducedmodulation(Buntingetal.,2015; Culleyetal.,2006;Dingetal.,2017;Edmandsetal.,2013;Lowesetal., 2017;Zureketal., 2014).Threegenesbelongtothesignaltransmis- sionclass(soluteCarrierfamily32member1[Slc32a1],bestrophin1 [Best1],andsodiumvoltage-gatedchannelalphasubunit9[Scn9a]),1 (homeodomaininteractingproteinkinase2[Hipk2])toproteinkinase class,and1(stannin[Snn])tothebitopicproteinclass.

Isoflurane-inducedchangesintheexpressionofsignaltransmission classgenescouldpotentiallybethemaincontributortothelong-term functionalconnectivitychangesobserved here.Slc32a1codesforthe vesicularinhibitoryaminoacidtransporter,whichisanintegralneu- ronal membrane protein involved in GABAand glycine uptakeinto synapticvesicles(McIntireetal.,1997).Theproteinisneededforef- ficientGABAtransmission,anditsreducedexpressionleadstoglobal inhibitiondeficiencies.Best1isacalcium-activatedanionchannelex-

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pressedinastrocytesthatactsasachloridetransporter,butalsoasa glutamatetransporter(OhandLee,2017).Interestingly,astrocyticglu- tamate transportationmayincrease duringBS anesthesia, leadingto diminishedcorticalinhibitionviacorticalinterneurons(Ferronetal., 2009).Notably,inourstudy,initialtreatmentwith1.8%isoﬂuranecon- centrationalsocausestheBSphenomenon.Finally,thescn9agenecodes fora voltage-dependentsodiumchannel (Klugbaueretal., 1995) in- volvedinthegenerationofactionpotentials(Renganathanetal.,2001).

Inourstudy,1monthaftertheisoﬂuranetreatment,slc32a1expres- sionwasdecreased0.2-fold,best1wasincreased1.1-fold,andscn9awas increased1.6-foldinthecortex.Thesegeneexpressionchangessuggest reducedglobalinhibitionandcorticalinhibition,andincreasedgenera- tionofactionpotentials,andthuspotentiallycontributetothechanges detectedinfunctionalconnectivityandinfMRIpowerinthecortexof isoﬂurane-treatedanimals.Interestingly,wealsofoundchangesinthe expressionofgapjunctiondelta2gene.Gapjunctiondelta2isinvolved in gapjunction-mediatedsignal transmission,andcontributes tothe generationof micromechanical-inducedburstingactivityorin home- ostaticplasticity(KroegerandAmzica,2007).

Othergenesnotrelatedtosignaltransmission,butthatcouldcon- tribute toisoflurane-mediated effects are Hipk2 andSnn. Hipk2en- codesaconservedserine/threoninekinaseandishighlyexpressedin thebrain(Kimetal.,1998;Wangetal.,2001).Hipk2-encodingkinases canactincaspase-mediatedsignalpathways,leadingtoneuronalapop- tosis(Doxakisetal.,2004).Increasedneuronalapoptosisinresponseto isofluraneanesthesiahasbeenreportedinmanycellandanimalstud- iesinvestigatingtheshort-termconsequencesofisofluraneanesthesia (Colonetal.,2017).The0.6-foldincreasedexpressionofHlpk2inthe presentstudysuggestsincreasedapoptoticactivity1month afterthe isofluranetreatment.Further,wefounda0.2-folddecreaseinthecor- ticalexpressionofSnn,whichcodesforaproteinthatmayhavearole in theselectivetoxicity oforganotins (Davidson etal.,2004). Possi- blemechanismsofHipk2andSnncontributingtothelong-termeffects ofisofluraneonfunctionalconnectivity,however,remaintobedeter- mined.

4.4. Physiologicandmethodologicconsiderations

As physiologic parameters were not measured during the initial isofluranetreatment,wecannotruleoutthepotentialeffectofrespi- ratorydepressioncausinghypoxiaontheresults.Weusedpractically identicalanestheticconditionsin ourpreviousstudies,however,and demonstratedthatanimalsareneitherhypoxicnorhypercarbicbased onbloodgasmeasurements(Jokivarsietal.,2009).Inadditiontoini- tialisofluranetreatment,weperformedourmeasurementsduringisoflu- rane anesthesia.As isofluranepotentiallyinduces slight hypercapnia andincreasesbasalbloodflow,carefulattentionwaspaidtoensurethat normalphysiologicconditionsweremaintainedasmuchaspossiblein eachanimal.Additionally,isofluranecanmaskintrinsicconnectivityby inducingBSactivity,whichwascarefullycontrolledbyobservingthe group-levelburstingparametersandanalyzingthedatawithandwith- outtheglobalsignalregression.

IntheMRImeasurements,weusedSE-EPIforfunctionalimaging.

AlthoughSE-EPIhashigherspatial speciﬁcity,itsuﬀersfrom poorer functionalcontrastcomparedwithgradientecho-EPI,whichcanlimit thesensitivityfordetectingfunctionalnetworks.

Thepresentstudywasconductedwithonlyasingleisofluranetreat- mentsessionusing aspecificconcentration(1.8%), afixed exposure time(3h),andasinglewaitingperiodbetweentheisofluranetreatment andthereadouts.Interestingly, Orsiniandco-workers (Orsiniet al., 2018) demonstrated increased corticalconnectivity at 1 month,but notafter2weeks,following0.5%isoflurane/medetomidineanesthesia.

Clearly,additionalstudiesareneededtoevaluatefunctionalchangesin responsetodifferentisofluraneconcentrations,exposuretimes,andvar- iousreadoutpointsafterthetreatment.Otherfuturedirectionsinvolve studyingtheagedependenceofanesthesiaeffects,astheinfluenceof

anesthesiaisofprimaryimportancetoearly-lifeinfantsandintheaged population(Colonetal.,2017).

MostofthesignificantfMRIconnectivitychangesweredetecteddur- inganesthesiawith2.0%isoflurane,althoughsimilartrendsofchanged connectivitywerealsoobservedduringanesthesiawith1.3%isoflurane.

AsmostofthefMRIstudiesareconductedataloweranesthesiadosing than2.0%,thechangesmaynothaveasigniﬁcanteﬀecton readout parameterswiththecurrentsamplesize.

DifferentpreparationstepsintheLFPgroupledto~70minlonger initialanesthesiadurationcomparedwiththefMRIgroup.Longerisoflu- raneexposureduringanimalpreparationbeforescanningcanalterfunc- tionalconnectivity(Magnusonetal.,2014).Therefore,slightdifferences betweentheLFPandfMRIconnectivityresultscouldarisefromdifferent preparationtimesbetweenthegroups.

The geneexpressionfindings werebased on onlyone technique, namelyRNA-seq.Although RNA-seqfindings areoftenconfirmedby qPCR,theRNA-seqtechniquebyitselfhaslowtechnicalvariabilityand isahighlyquantitativetechnique(Wangetal.,2009).RNA-seqresults provide abroaderviewofthegeneexpression,however,whencom- paredwithqPCR.Therefore,moreweightshouldbegiventotheob- servedgeneclassesinsteadofindividualgenes.Also,itshouldbenoted that mRNAexpressiondoesnot directlyimply change inprotein ex- pressionascompensationmechanismsmayalsooccur(El-Brolosyand Stainier,2017).

5. Conclusions

Ourfunctionalconnectivity,electrophysiological,andgeneexpres- sionfindingsindicatethatasinglelong1.8%isofluraneexposure,deep enoughtocauseBSactivity,haslong-termeffectsonbrainfunctionthat lastatleast1month.Thiswassupportedbythefindingsofincreased hippocampal-corticalandthalamo-corticalfunctionalconnectivity,in- creasedcorticalfMRIsignalpower,expressionchangesin genesthat promoteneuroplasticity,andneuronalsignaltransmissionmechanisms.

Duringthemeasurements,nochangeswereseeninestablishedmeasures ofthedepthofanesthesia,theburstoccurrencerate,suppressionduration,orburstamplitude,andthereforetheobservedalterationsinbrain functionlikely reflectneuralnetworkplasticity ratherthanachange inthedepthofanesthesiaexperiencedbytheanimalduringthemea- surements.Inadditiontoprovidinginsightsintocellular-levelandfunc- tionalisoflurane-inducedchangesinthebrain,ourresultsareimportant forpreclinicallaboratoriesusingisofluraneanesthesiaandconducting follow-upfunctionalconnectivitystudies,astheobservedeffectsmay influencethestudydesign.

Creditauthorstatement

Petteri Stenroos: Conceptualization, Methodology, Investigation, Writing-OriginalDraft,Writing-Review&Editing,Visualization

Tiina Pirttimäki:Conceptualization, Methodology, Investigation, Writing-Review&Editing

JaakkoPaasonen:Investigation,Writing-OriginalDraft,Writing- Review&Editing,Supervision

EkaterinaPaasonen:Software,Formalanalysis RaimoASalo:Software,Formalanalysis HennariikkaKoivisto:Investigation TeemuNatunen:Methodology PetraMäkinen:Investigation

TeemuKuulasmaa:Software,Formalanalysis

MikkoHiltunen:Methodology,Resources,Writing-Review&Edit- ing,Fundingacquisition

Heikki Tanila: Conceptualization, Methodology, Validation, Re- sources,Writing-Review&Editing,Supervision,Fundingacquisition

OlliGröhn:Conceptualization,Methodology,Validation,Resources, Writing-OriginalDraft,Writing-Review&Editing,Supervision,Project administration,Fundingacquisition

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DeclarationofCompetingInterest None.

Acknowledgments

WeliketothankMaaritPulkkinenfortechnicalassistancein ani- malpreparations.ThisstudywassupportedbyFinnishFunctionalGe- nomicsCentre,UniversityofTurku,ÅboAkademiUniversityandBio- centerFinland.MRIwasdoneinBiomedicalImagingUnitcorefacility, UniversityofEastern Finland,Finland.Geneexpressiondataanalysis wascarriedoutwiththesupportofUEFBioinformaticsCenter,Univer- sityofEasternFinland,Finland.ThisworkwassupportedbyAcademy ofFinland(grant numbers298007,307866);Sigrid JuséliusFounda- tion;theStrategicNeuroscience FundingoftheUniversityof Eastern Finland;andtheDoctoralPrograminMolecularMedicineofUniversity ofEasternFinland.Thefundingsourceshadnoroleinstudydesignor inthecollection,analysis,orinterpretationofdata.

Dataandcodeavailabilitystatements

Original data of this study are available at Mendeley Data (http://dx.doi.org/10.17632/wxftdp4n6x.1).In-housemodiﬁedscripts areavailablefromthecorrespondingauthorbyemail.

Supplementarymaterials

Supplementarymaterialassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.neuroimage.2021.117987.

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