The effect of parvalbumin deficiency on the acoustic startle response and prepulse inhibition in mice

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The

effect

of

parvalbumin

deﬁciency

on

the

acoustic

startle

response

and

prepulse

inhibition

in

mice

Jiˇrí

Popeláˇr

a,∗

_,

_Natalia

_Rybalko

a

_,

_Jana

_Burianová

a

_,

_Beat

_Schwaller

b

_,

_Josef

_Syka

a

a_Institute_of_Experimental_Medicine,_Academy_of_Sciences_of_the_Czech_Republic,_Department_of_Auditory_{Neuroscience,}₁₄₂₂₀_Prague,_Czech_Republic

b_Anatomy,_Department_of_Medicine,_University_of_Fribourg,_CH-1700_Fribourg,_Switzerland

h

i

g

h

l

i

g

h

t

s

•PV−/−micehadsmallerASRamplitudesinresponsetorelativelyweakstartlingstimuli(80–90dBSPL)incomparisonwithPV+/+mice.

•PPIoftheASRinPV−/−micewaslesseffectivethaninPV+/+mice.

•MeanABRaudiogramswerefoundtobesimilarinbothgenotypes.

Thestrengthoftheacousticstartleresponse(ASR)toshortburstsofbroadbandnoiseortonepips(4,8

and16kHz)andtheprepulseinhibition(PPI)oftheASRelicitedbyprepulsetones(4,8and16kHz)were

measuredinparvalbumin-deﬁcient(PV−/−)miceandinage-matchedPV+/+miceascontrols.Hearing

thresholdsasdeterminedfromrecordingsofauditorybrainstemresponseswerefoundtobesimilarin

bothgenotypes.TheASRstobroadbandnoiseandtonesoflowandmiddlefrequencieswerestronger

thantheASRsinresponsetohigh-frequencytonesinbothgroups.InPV−/−mice,weobservedsmaller

ASRamplitudesinresponsetorelativelyweakstartlingstimuli(80–90dBsoundpressurelevel(SPL))of

eitherbroadbandnoiseor8-kHztonescomparedtothoserecordedinPV+/+mice.Forthesestartling

stimuli,PV−/−micehadhigherASRthresholdsandlongerASRlatencies.PPIoftheASRinPV−/−mice

waslesseffectivethaninPV+/+mice,foralltestedprepulsefrequencies(4,8or16kHz)at70dBSPL.

OurﬁndingsdemonstratenoeffectofPVdeﬁciencyonhearingthresholdsinPV−/−mice.However,the

frequency-specificdifferencesintheASRandthesignificantreductionofPPIofASRlikelyreflectspecific

changesofneuronalcircuits,mainlyinhibitory,intheauditorycentersinPV-deﬁcientmice.

.

1. Introduction

Parvalbumin(PV)belongstoalargefamilyofEF-hand calcium-bindingproteins,whichcomprisesmorethan240membersinman [21]. Studiesoncalcium-bindingproteinfunctionsindicatethat theseproteinsareessentialinCa2+_homeostasis_and_for_the

modu-lationofshort-livedchangesintheintracellularCa2+_{concentration}

calledCa2+_transients._For_PV,_this_is_the_case_in_fast-twitch

mus-cles[23]andinaspeciﬁcsubpopulationofneurons[2],wherePVis implicatedinthesubtleregulationandtimingofCa2+_signals

pre-andpostsynaptically.Inthebrain,PVisalmostexclusivelypresent

∗ Correspondingauthorat:InstituteofExperimentalMedicineASCR,Department

ofAuditoryNeuroscience,Videnska1083,14220Prague4,CzechRepublic.

Tel.:+420241062689;fax:+420241062787.

E-mailaddresses:[email protected](J.Popeláˇr),[email protected]

(N.Rybalko),[email protected](J.Burianová),[email protected]

(B.Schwaller),[email protected](J.Syka).

insubpopulationsofinhibitoryGABAergicinterneuronsindifferent brainregionsincludingtheneocortex,cerebellum,hippocampus andthereticularnucleusofthethalamus[2–4,18].Intheauditory systemPVisexpressedincochlearhaircells[11]andinglobular bushycells’endingsinthelargecalyxofHeldontheprincipalcells inthemedialnucleusofthetrapezoidbody[8].Alargenumberof PV-immunoreactiveneuronsarepresentinallsubnucleiofthe infe-riorcolliculus,PV-positiveneuronsarescatteredthroughoutlayers II–VIintheauditorycortexandsparsesmall,ovalPV-positive neu-ronsarescatteredinboththedorsalandventraldivisionsofthe medialgeniculatebody[19].Inthecaseofthemedialgeniculate body thedistribution of GABA-ergic neuronsis highly species-speciﬁc:theproportionrangesfrom<1%inthebatandrattoabout 25%inthecatandmonkey[30].However,onlylimited informa-tionexistsontheroleofPVintheauditorysystem.Severalrecent studieshavereportedage-relatedandregion-speciﬁcchangesin theexpressionofPV-positiveneuronsinavarietyofmammalian species:anage-relateddeclineofcalciumbindingprotein-positive neurons in thedorsal cochlear nucleusof CBA/CaJ mice[13],a

Published in 1HXURVFLHQFH/HWWHUV±

which should be cited to refer to this work.

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decreaseinthepercentageofPV-expressingneuronsinthe superfi-ciallayersoftheauditorycortexofC57Bl/6mice[17]oranincrease ofPV-positiveneuronsintheinferiorcolliculusofoldLongEvans ratsandapronounceddeclineinthenumberofPV-positiveneurons intheauditorycortexofagedFischer344rats[19].Theassessment ofthefunctionofPVinmicehasbeenfacilitatedbyusinggenetically modifiedmicedeficientforthisprotein[23].Theresultsobtained ininvitrostudiesusingPV−/−micesuggestthatPVhasevolvedas afunctionallydistinct,physiologicallyrelevantmodulatorof intra-cellularCa2+_transients_[22,24,25]_._Behavioral_tests_performed_in

PV−/−micerevealedsubtlealterationsoflocomotorfunctionin comparisonwithPV+/+mice,characterizedbyaslightlyincreased motoractivity,decreasedexploratoryactivityandincreased micro-linearityofmovements[7].However,thefunctionalconsequences ofPVdeﬁciency interms ofauditoryperceptionhave notbeen studieduntilnow.

In thepresent study, PV−/− micewere used toinvestigate, whetherthelossofPVhasaneffectonauditorybehavior,inwhich both sensoryand motor components play importantroles. The strengthoftheacousticstartlereflex(ASR)andtheauditory pre-pulseinhibition(PPI)wereevaluatedinPV−/−miceandcompared withthoseobservedinage-matchedPV+/+mice.TheASR,defined asatransientmotorresponsetoanintense,unexpectedstimulus, wasusedasanindicatorofthebehavioralresponsivenesstoloud sounds.TheassessmentofauditoryPPI,i.e.thesuppressionofthe ASRbya prepulseprecedingthestartlingstimulus,wasusedas anindicatorofpossiblechangesininhibitoryfunction.PPIis con-sideredtobeaformofsensorimotorgatingthatreflectsabasic inhibitoryprocessthatregulatessensoryinputtothebrain[28].In addition,measurementsoftheauditorythreshold,whichcan influ-enceASRandPPI,wereperformedbyrecordingauditorybrainstem responses.

2. Materialsandmethods

2.1. Animals

Parvalbumin-deﬁcientmice(PV−/−)weregeneratedby homol-ogousrecombination[23]andbackcrossedtoC57BL/6micefor>10 generationsand areconsideredtobecongenictoC57Bl/6mice [18].PV−/−mice(n=9,meanbodyweight29.0±1.6g)obtained directlyfromtheanimalfacilityoftheUniversityofFribourgwere genotypedbyPCR[23]tovalidatetheinactivationofthePvalbgene. Animalsweretestedatﬁvemonthsofageandtheresultswere com-paredwithage-matchedC57BL/6wildtypemales(PV+/+;n=10, meanbodyweight30.1±1.4g).

Micewerehousedunderstandard conditions ona 12h/12h light/darkcycle.Thecareanduseofanimalsandall experimen-talprocedureswereperformedincompliancewiththeguidelines oftheEthicalCommitteeoftheInstituteofExperimentalMedicine, AcademyofSciencesoftheCzechRepublic,andfollowedthe Euro-peanCommunityDirective86/609/EEC.

2.2. Behavioraltests

All behavioral tests were performed in a ventilated sound-attenuatedchamber(CoulbournHabitest,modelE10-21)located inasoundproofroom.Duringthetestingprocedure,micewere conﬁned to a small wire mesh cage (85×45×45mm) on a motion-sensitiveplatform.Thewhole-bodystartleresponseswere detectedandtransducedbyapiezoelectricaccelerometer (Coul-bournE45-11B).The ampliﬁedvoltage signalwasacquired and processed using a RP2.1 enhanced real-time processor (Tucker DavisTechnologiessystemIII,Alachua,FL).Thestartleresponses were evaluated in a 100-ms window beginning at the onset

of the startle stimulus. Acoustic startle stimuli (tone pips or noise bursts) and prepulse stimuli (tone pips) were generated bytheRP2.1enhanced real-timeprocessor andpresented viaa loudspeaker (SEAS, 29AF/W) placed 12cm above the platform. ASRsto 4,8 and 16kHz tone pipsand broadband noise bursts (witharelativelyﬂatfrequencyspectrumbetween2and35kHz) of 50-ms duration and varying intensity levels were recorded. In the ASR experiments, each session contained 9 trial types: a baseline trial without any stimulus and 8 startle stimuli of differentintensities(50–120dBSPLwithastepsizeof10dB) pre-sentedinarandomorderatintervalsof10–30s;eachtrialtype waspresentedtentimes.TheASRthreshold,theASRamplitude and the latency of the ASR to 110dB SPL sound were ana-lyzed.

InthePPItesting,threetrialtypeswereused:abaselinetrial withoutanystimulus,astartlingpulsealone(115dBSPL broad-bandnoiseburstof50ms),andacombinationofthestartlepulse and50-msprepulsetonesof70dBSPL,atfrequenciesof4,8and 16kHz.Theintervalbetweentheprepulseandstartlingstimuliwas 50ms.Alltrialtypeswerepresented10timesinpseudo-random order separatedby 15–30s. Theefﬁcacyof thePPI of ASRwas expressedas:PPI%=(amplitudeoftheASRsuppressedbythe pre-pulsetone/amplitudeoftheASRalone)×100.Thus,aPPIof100% meansnoASRsuppression,aPPIof0%correspondstocomplete suppressionoftheASR.

2.3. Auditorybrainstemresponserecordings

Thehearing thresholdin micewas assessedon thebasis of auditorybrainstemresponserecordingsusingsubcutaneous nee-dleelectrodesplacedonthevertex (anactiveelectrode) andin the neck muscles (ground and reference electrodes). Auditory brainstemresponseswereelicitedusingshort-tonebursts(3-ms duration,1ms rise/falltimes,frequencyrange 4–32kHz) gener-atedwithaRP2.1enhancedreal-timeprocessor.Acousticstimuli weredeliveredinfree-fieldconditionsviaatwo-wayloudspeaker system[Jamo®woofer(Denmark)andSEAS®T25CF002tweeter (Norway)]placed70cminfrontoftheanimal’shead.The acous-ticsystemwascalibratedwithaBruel&Kjaer®4939microphone, aZC0020 preamplifierand aB&K 2231SoundLevelMeter.The frequency–responsecurveof thissystemwasrelativelyflatand variedby less than±9dBbetween0.15 and 40kHz. Thesignal fromtheelectrodewasamplified10,000-times,band-passfiltered overtherangeof300Hz–3kHz,processedwithaRX5-2Pentusa BaseStation(TuckerDavisTechnologiessystemIII,Alachua,FL)and analyzedusingBioSig software.Theresponse thresholdtoeach frequencywasdeterminedastheminimaltoneintensitythatstill evokedanoticeablepotentialpeakintheexpectedtimewindow oftherecordedsignal.

2.4. Statisticalanalysis

ThedifferencesbetweenhearingthresholdsandASRamplitudes inPV−/−miceandPV+/+miceweretestedusingtwo-wayANOVA withtheBonferronipost-test.ThedifferencesbetweenASR thresh-olds,ASRlatenciesandPPIofASRatindividualfrequencieswere testedusinganunpairedt-test.

3. Results

3.1. Hearingthresholds

HearingthresholdsinPV−/−micetendedtobeslightlylower (onaverageby5–10dB)thanthethresholdsinPV+/+mice,butthe differenceswerenotsigniﬁcant(p>0.05,two-wayANOVA, Bon-ferronipost-test).Theaveragehearingthresholdsin5-month-old

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Fig.1.MeanASRamplitudevs.soundintensityfunctionsinPV−/−andPV+/+miceobtainedfortonepipsof4,8,and16kHz,aswellasforbroadbandnoisebursts.Bars

represent±SEM;“B”-baselinetrial;*p<0.05,**p<0.01,two-wayANOVAwiththeBonferronipost-test.

PV−/−andPV+/+miceareshowninSupplementarymaterialinFig. S1.

3.2. Acousticstartlereﬂex(ASR)

ThedependenceoftheASRamplitudeonthestartlingstimulus intensityforPV−/−andPV+/+miceisshowninFig.1.

TheASRstobroadbandnoiseandtonesof4and8kHz(Fig.1A, BandD)weregenerallystrongerthantheASRsto16-kHztones (Fig.1C)inallmice.ThedifferencesbetweentheASRsinPV−/− and PV+/+ micewereclearly evidentin response tobroadband noiseandtotonesof8kHz:theASRamplitudesevokedbythese startlingstimuliweresmallerinPV−/−thaninPV+/+mice(Fig.1B andD),butsigniﬁcantdifferenceswereonlyobservedat80and 90dBSPL(p<0.05andp<0.01,respectively,two-wayANOVAwith theBonferronipost-test).Inaddition,PV−/−micehadhigherASR thresholdsthandidPV+/+animals,butdifferencesweresigniﬁcant onlyat8kHz(78.9±6.0dBSPLvs.68.0±4.7dBSPL,resp.,p<0.01, unpairedt-test)and16kHz(87.5±4.2dBSPLvs.78.5±6.6dBSPL, resp.;p<0.05,unpairedt-test).

ThelatenciesoftheﬁrstpositivewaveoftheASR evokedby 110-dBstartlestimuliwereanalyzed(Table1).PV−/−mice exhib-itedlongerASRlatenciesincomparisontoPV+/+mice.Despitethe factthattheiramplitudes,evokedby110dBSPLstartlestimuli, weresimilar,thedifferencesinlatenciesweresigniﬁcantat4and

Table1

AveragelatenciesoftheASRinPV−/−andPV+/+mice(mean±SEM).*p<0.05,

**p<0.01,unpairedt-test. Startlestimulus(110dBSPL) PV−/− PV+/+ Broadbandnoise 25.0±0.6ms ns 25.1±0.6ms 4kHz 26.2±0.5ms ** 25.3±0.4ms 8kHz 25.9±0.9ms * 24.7±0.7ms 16kHz 26.4±0.7ms ns 25.5±1.4ms

8kHz(*p<0.05and**p<0.01,resp.,unpairedt-test).TheASR laten-ciesevokedbybroadbandnoiseweresimilarinbothexperimental groupsofmice.

3.3. Prepulseinhibition(PPI)ofASR

TheeffectofmodifyingtheASRamplitudesbyapreceding stim-uluswastestedusingPPIofASRinmiceofbothgenotypes.Fig.2 showsthechangesintheaveragerelativeASRamplitudesevoked by115dBSPLbroadbandnoisestimuli,usingprepulsestimuliat4, 8and16kHzatanintensityof70dBSPLinPV−/−andPV+/+mice; alowerrelativeASRamplitudecorrespondstostrongerPPI(from noinhibitionat100%tocompleteASRinhibitionat0%).Theresults documentasigniﬁcantlylesseffectivePPIinPV−/−micethanin PV+/+controlsatalltestedPPIfrequencies(*p<0.05,***p<0.001, unpairedt-test).

Fig.2.TheefﬁcacyofPPIofASRinPV−/−andPV+/+mice.TheefﬁcacyofthePPIof

ASRwasexpressedas:PPI=(ASRamplitudesevokedby115dBSPLbroadbandnoise

stimuliandsuppressedbytheprepulsetoneat4,8or16kHzatanintensityof70dB

SPL/amplitudeoftheASRalone)×100%.APPIof0%correspondstocompleteASR

suppression.*p<0.05,***p<0.001,unpairedt-test),barsrepresent±SEM.

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4. Discussion

TheresultsofourstudydemonstratethattheabsenceofPVin PV−/−micealteredASRparametersandthatthesemiceshowed signiﬁcantlylessefﬁcientprepulseinhibitionoftheASRin com-parisontoPV+/+micewithnormalPVlevels.Theaveragehearing thresholdsinPV−/−micewere,however,comparablewiththose foundinPV+/+mice.Thecalcium-bindingproteinPVispresentin boththeinnerandouterhaircellsinthecochlea,anditsroleisto modulateCa2+_entry_to_the_hair_cell_cytoplasm_during

mechano-electricaltransductionandafferentandefferentsynapticactivity [10].DuetothesimilarityofthehearingthresholdsinPV−/−and PV+/+mice,theroleofPVinCa2+_buffering_does_not_seem_to_be

essentialforthesubject’shearingsensitivitynearthethreshold. TheASR,asanunconditionedreflexreactiontoanabruptloud sound, is represented by a short neural circuit comprising the posteroventralcochlear nucleus, oneor more nucleiof the lat-erallemniscus, the caudal pontine reticularnucleus and spinal interneurons and motor neurons [5,15]. In spite of its relative simplicity,theASR shows severalformsof behavioralplasticity (habituation,facilitation,prepulseinhibitionandmodificationby priorassociativelearning)thatreflecttheprocessingofacoustic informationathigherlevelsoftheauditorysystemthanthe neu-ralcircuitoftheASRitself[14,15,20].Thesefeaturesofthestartle reflexallowthestudyofnotonlytheprocessingofauditorystimuli, butalsoofcomplexbrainfunction,includingmovementcontrolor cognitivedisorders.

OurresultsindicatethattheabsenceofPVinduces “abnormal-ities”intheneuralprocessesthatfinallyresultinchangesofthe startlereflexformation.ThesignificantlylongerASRlatency,found inourstudyinPV-deficientmicecomparedwithPV+/+mice,might berelatedtotheprolongationofthetimerequiredtoattainpeak forceand the increasedforce produced duringa singlemuscle twitch[23],oritcouldbelinkedtoamild impairmentin loco-motorcoordinationthathasbeenobservedinPV−/−mice[7].The mostdistinctdifferencesinASRparameters,i.e.significantlyhigher ASRthresholds,lowerASRamplitudesandlongerASRlatenciesin PV−/−miceincomparisonwithPV+/+mice,wereobservedusing 8-kHztones.Thisisnotsurprisinggiventhatthehearing sensi-tivitywashighestandthereflexresponsewasmorepronounced (especiallyincomparisonwith16kHz)inbothgenotypesatthis frequency(Fig.S1,Fig.1).TheseresultssuggestthatASRdifferences reflectnotonlyparvalbumindeficiencyintheskeletalmuscles,but mostlikelyalsospecificchangesintheauditoryneuronalcircuits. ThePPIofASRrepresentsabasicinhibitoryprocessregulating sensoryinputs,andthemagnitudeofPPIcanserveasameasure forthebehavioralsalienceofperceptually-relevantstimuli[9,29]. Previousanimalstudieshaveshown thatforPPIofASR,several pathwaysareimplicatedincludingdirectglutamatergicprojections fromthecochlearnuclei,inhibitorycholinergicprojectionsfrom thepedunculopontinetegmentalnucleusandindirectinputsfrom thesuperiorandinferiorcolliculi[15].Electrophysiological recor-dingsofcortical,hippocampalandcerebellarneuronalactivityin PV−/−miceinvitroshowedthattheabsenceofPVina subpopu-lationofGABA-ergicinterneuronsresultedinmodificationofthe dynamicsofinhibitorycontrol (forreviews,see[22]), enhance-mentoftheseverityofpharmacologically-inducedseizures[25], alterationsofPurkinjecell-firing[26],andmodulationofthe fir-ingpropertiesofthereticularthalamicnucleusburstingneurons [1].Fast-spikingPV-positiveinhibitorycells,whichrepresentthe largestinhibitorysubpopulationintheauditorycortexofthecat, areinvolvedinthesharpeningoffrequencyandintensitytuning andtheshapingofspectralmodulationpreferences[27,31]. How-ever,little is known sofar about the influence of the missing parvalbuminonneuronalactivityinthecentralauditorynucleiin PV−/−mice.Theresultsofanimalstudieshavesuggestedthatfor

thePPIofASR,thecochlearnucleus,thesuperiorandinferior col-liculiandthepedunculopontinetegmentalnucleusareimportant [15,29],andPPIcanbemodulatedbytheauditorycortex, amyg-dala,hippocampusandstriatum[29].Ourdataclearlydemonstrate thattheeffectofPPIonASRismuchweakerinPV−/−micethan incontrols.Thiscouldbelinkedtothedecreaseddensityof PV-positiveinhibitoryinterneurons,possiblyastheresultofreduced PVexpressionlevels,intheauditorynucleiofsomemouseandrat strainsduringaging[13,19].Inline,indystonichamsters show-ingatransientdecreaseinPV-positiveneuronsand/orPVlevelsat approximately30daysofage,ASRisdecreased[11].Thelow efﬁ-ciencyofPPIinPV−/−micecouldbealsorelatedtoalteredfunction ofthecortico-tectalpathways,whichareunderthestronginﬂuence ofparvalbumininterneuronsintheauditorycortex[6].

Atthebehaviorallevel,thetestingofPPIofASRhasbeenused fordetectinginhibitoryalterationsinvarioussensori-motor sys-tems.AdeﬁciencyoreventheabsenceofPPIofASRwasobserved inmicewithGABAAreceptor␥2subunitsremovedfromthe

PV-positiveneurons[16],inGAD65knockoutmice[12]andindystonic hamsters[11].

Inthepresentstudy,areducedPPIwasobservedinPV−/−mice atallprepulsestimulusfrequencies,i.e.4,8and16kHz.Becausethe hearingthresholdsweresimilarinbothgenotypes,theobserved differenceswithrespecttoauditorybehavior(ASRandPPIofASR) arenotdirectlyrelatedtoperipheralhearingsensitivity.Ourdata supporttheideathattheabsenceofPVinthecentralauditorynuclei isresponsibleforchangesintheinhibitorysystemresultinginthe reductionofPPIofASR.Forinvestigatingspeciﬁcchangesinthe centralauditorysystemofPV−/−mice,electrophysiological exper-imentsandmoresophisticatedbehavioraltestsusingconditioning proceduresshouldbeperformed.

Inconclusion,theresultsof ourstudydemonstratenoeffect ofPVdeficiencyontheauditoryperiphery,resultinginunaltered hearingthresholds.Thefrequency-specificdifferencesintheASR andthesignificantreductionofPPIofASR inPV-deficientmice likelyreflect specificchangesof neuronalcircuits and/orcircuit function,mainlyinhibitory,intheauditorycentersinPV-deficient mice.

Conﬂictofinterest

Theauthorshavedeclaredthatnoconﬂictofinterestexists.

Acknowledgments

This work was supported by the Grant P304/12/1342 from theGrant Agencyof theCzechRepublic and bytheprojectOP RDICZ.1.05/1.1.00/02.0109“BiotechnologyandBiomedicine Cen-teroftheAcademyofSciencesandCharlesUniversityinVestec (BIOCEV)”, OperationalProgram Research and Development for InnovationsbytheMinistryofEducation,YouthandSportsofthe CzechRepublicandtheEuropeanRegionalDevelopmentFund.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version

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