Mosienko, V., Chang, AJ, Alenina, N., Teschemacher, AG, & Kasparov, S.

15 Pages·2017·2.1 MB·English

Mosienko, V., Chang, A. J., Alenina, N., Teschemacher, A. G., &

Kasparov, S. (2017). Rodents and humans are able to detect the
odour of L-Lactate. PLoS ONE, 12(5), [e0178478].
https://doi.org/10.1371/journal.pone.0178478
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RESEARCHARTICLE
Rodents and humans are able to detect the
odour of L-Lactate
ValentinaMosienko1,AndyJ.Chang2¤,NataliaAlenina3,AnjaG.Teschemacher1,
SergeyKasparov1,4*
1 SchoolofPhysiology,PharmacologyandNeuroscience,UniversityofBristol,Bristol,UnitedKingdom,
2 DepartmentofBiochemistryandHowardHughesMedicalInstitute,StanfordUniversitySchoolofMedicine,
Stanford,UnitedStatesofAmerica,3 Max-Delbru¨ck-CenterforMolecularMedicine(MDC)Berlin-Buch,
Berlin,Germany,4 BalticFederalUniversity,Kalinigrad,RussianFederation
¤ Currentaddress:DepartmentofPhysiologyandCardiovascularResearchInstitute,UniversityofCalifornia,
SanFrancisco,UnitedStatesofAmerica
*[email protected]
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Abstract
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L-Lactate(LL)isanessentialcellularmetabolitewhichcanbeusedtogenerateenergy.In
addition,accumulatingevidencesuggeststhatLLisusedforinter-cellularsignalling.Some
LL-sensitivereceptorshavebeenidentifiedbutwerecentlyproposedthattheremaybeyet
anotherunknownG-proteincoupledreceptor(GPCR)sensitivetoLLinthebrain.Olfactory
OPENACCESS
receptors(ORs)representthelargestfamilyofGPCRsandsomeofthemareexpressed
Citation:MosienkoV,ChangAJ,AleninaN,
outsidetheolfactorysystem,includingbrain,makingtheminterestingcandidatesfornon-
TeschemacherAG,KasparovS(2017)Rodents
olfactoryLLsignalling.Oneofthe“ectopically”expressedORs,Olfr78inmice(Olr59inrats
andhumansareabletodetecttheodourofL-
Lactate.PLoSONE12(5):e0178478.https://doi. andOR51E2inhumans),reportedlycanbeactivatedbyLL.Thisimpliesthatbothrodents
org/10.1371/journal.pone.0178478 andhumansshouldbeabletodetecttheLLodour.Surprisingly,thishasneverbeendemon-
Editor:JohannesReisert,MonellChemicalSenses strated.HereweshowthatmicecandetecttheodourofLLinodourdetectionandhabitua-
Center,UNITEDSTATES tion-dishabituationtasks,anddiscriminateitfrompeppermintandvanillaodours.Behaviour
Received:January16,2017 oftheOlfr78nullmiceandwildtypemiceinodourdetectiontaskwasnotdifferent,indicating
thatrodentsareequippedwithmorethanoneLL-sensitiveOR.Ratswerealsoabletouse
Accepted:May12,2017
thesmellofLLasacueinanodour-rewardassociativelearningtask.Whenpresentedto
Published:May25,2017
humans,morethan90%ofparticipantsdetectedasmellofLLinsolution.Interestingly,LL
Copyright:©2017Mosienkoetal.Thisisanopen
wasperceiveddifferentlythanacetateorpropionate—LLwaspreferentiallyreportedasa
accessarticledistributedunderthetermsofthe
pleasantsweetscentwhileacetateandpropionatewereperceivedasrepulsivesour/acid
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and smells.SubjectiveperceptionofLLsmellwasdifferentinmenandwomen.Takentogether,
reproductioninanymedium,providedtheoriginal ourdatademonstratethatbothrodentsandhumansareabletodetecttheodourofLL.
authorandsourcearecredited.
Moreover,inmice,LLperceptionisnotpurelymediatedbyOlfr78.DiscoveryoffurtherLL-
DataAvailabilityStatement:Allrelevantdataare sensitiveORmightshedthelightontheircontributiontoLLsignallinginthebody.
withinthepaper.
Funding:ThisworkwassupportedbyBBSRC:BB/
L019396/1,BB/K009192/1;andMRCMR/
L020661/1.Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto
Introduction
publish,orpreparationofthemanuscript.
L-Lactate(LL)isproducedbyallcellsinthebody.Inthecentralnervoussystem(CNS)both
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. neuronsandastrocytesproduceanduseLLbuttheabilitytostoreglycogenmakesastrocytesa
PLOSONE|https://doi.org/10.1371/journal.pone.0178478 May25,2017 1/14
L-Lactateodourperceptioninrodentsandhumans
particularlyimportantsourceofLLduringhighenergydemand[1,2],leadingtothehypothe-
sisofastrocyte-to-neuronlactateshuttling.Accordingtothishypothesis,astrocytesproduce
andreleaseLLwhichcanbetakenupandconvertedtoglucosebyneuronsduringperiodsof
neuronalactivation[3,4].IthasrecentlybecomeevidentthatLLmayalsoactasasignalling
moleculeviacognateG-proteincoupledreceptors(GPCR)[5].Oneofthese,knownasHCA1
(previouslyGPR81),hasnowbeenformallyrecognisedasareceptorforLL[6,7].HCA1is
mainlyexpressedinadiposetissue,butlowlevelswerealsoreportedinvariouspartsofthe
brain[8–10].SincemultiplestudiessuggestthatactivationofHCA1requiresveryhighLLcon-
centrations[8,9],itisunlikelythatithasanyfunctioninthebrainunderphysiologicalcondi-
tions.OurrecentresultspointedtotheexistenceofanotherLL-sensitiveG-coupledGPCRin
s
rodentbrain.ActingonthisputativeGPCR,LLevokesnoradrenalinereleaseandnoradrener-
gicneurondepolarisationinthelocuscoeruleus[11].TheseeffectsofLLareconcentration-
dependent,blockedbyD-Lactate,anddonotrequireLLentryintotheneuron.Identification
ofthisLL-sensitiveGPCRisstillpending,raisinginteresttoanynovelGPCRwhichcanpoten-
tiallymediateLLeffects.Inhumans,thereareabout800GPCRs,approximatelyhalfofwhich
mediateolfaction(http://www.guidetopharmacology.org).Activationofolfactoryreceptors
(ORs)leadstocAMPaccumulation,similarlytoG-coupledGPCRs.MostoftheORsare
s
exclusivelyexpressedinthesensoryneuronsofthemainolfactoryepitheliumbutectopic
expressionofORshasalsobeenreported.Forexample,intestis,ORsarethoughttoplayarole
inspermchemotaxis[12,13]buttheirrolesinothertissues,includingthebrain[14,15],are
completelyunknown.OneoftheseectopicallyexpressedORsisthemouseolfactoryreceptor
78(Olfr78)whichhasratandhumanorthologues(Olr59andOR51E2,respectively[16,17]).
Inthemouse,Olfr78wasdetectedinthebrainstemareapostremaandnucleustractussolitar-
ius[16]andlaterfoundinsmoothmusclecellsinsmallbloodvesselsinavarietyoftissues[18,
19].Inaddition,itisalsoexpressedintypeIglomuscellsofthecarotidbody[20],prostate
gland[21],colon,ovaries,testis,pancreas,andplacenta[22].HumanOR51E2andmouse
Olfr78dose-dependentlyrespondedtoacetateandpropionate(Olfr78:EC =2.35mMfor
50
acetateand0.92mMforpropionate;OR51E2:EC =2.93mMforacetateand2.16mMfor
50
propionate[18]),andLL(Olfr78:EC =4mM[20]).Thewidedistributionofthisreceptor
50
impliesitsroleinvitalphysiologicalprocessesotherthanolfaction.Inthekidney,Olfr78was
showntoregulateglomerularfiltrationrate,reninsecretion,andbloodpressure[18,19]while,
inthecarotidbody,itwasproposedtoplayaroleinhypoxiadetection[20].
PresenceofOlfr78intheolfactorysystemanditssensitivitytoLLsuggeststhatrodentsand
humansshouldbeabletodetectthesmellofLL.Surprisingly,thishasneverbeenconvincingly
documented.Moreover,inthePubChemdatabase,informationaboutwhetherLLhasasmell
iscontroversialwiththemostrecententrybeing“odourless”(https://pubchem.ncbi.nlm.nih.
gov/compound/612#section=Top).
Inthepresentstudy,weassessedwhetherrodentsandhumansare,infact,abletodetectan
odourofLL,andtheimportanceofOlfr78inLLdetection.Weshowthatmice,ratsand
humansareabletosmellLL.SinceOlfr78knockoutmicewereneverthelessabletodetectLL,
theremustbemorethanoneLL-sensitiveORinthatspecies.IdentificationofalternativeLL
signallingmechanismsmayshedlightontoLLtransmissionpathwaysoutsidetheolfactory
system.
Materialandmethods
Animals
AllproceduresinwildtypemicewereconductedinaccordancetotheguidelinesfromDirec-
tive2010/63/EUoftheEuropeanParliamentontheprotectionofanimalsusedforscientific
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L-Lactateodourperceptioninrodentsandhumans
purposes,andtheexperimentsperformedinthisstudywereapprovedbytheSenateofBerlin
EthicsCommittee(Landesamtfu¨rGesundheitundSozialesBerlin(LAGESO);studynumber
G0300/13).TheexperimentsinratswereperformedaccordingtotheUKHomeOffice’sAni-
mals(ScientificProcedures)Act,1986andapprovedbytheUniversityofBristolethicsreview
committee(protocolnumber30/2875).ExperimentsperformedusingtheOlfr78null/reporter
mouselinewereapprovedbytheInstitutionalAnimalCareandUseCommittee(IACUC)at
theStanfordUniversitySchoolofMedicine(protocolnumber19968).
Animalmaintenance
3and8maleC57Bl/6micewereusedforhabituation-dishabituationandodourdetection
tasks,respectively.7homozygousOlfr78null[23]and9wildtypelittermatecontrolmiceona
mixed129P2/OlaHsdandC57Bl/6Jbackgroundwereusedforanodourdetectiontask.All
miceweremale,5–9monthsofage,group-housed,andmaintainedunderanormallight/dark
cycle,withfreeaccesstofoodandwater.
12maleListerHoodedratsweighingbetween200and240g(Harlan)wereusedinan
odour-rewardassociativelearningtask.Allratswerehandledandweigheddaily,andmain-
tainedonareversed12-hourlight-darkcyclewithfreeaccesstofoodandwater.7daysafter
thearrivalanimalswereputonarestricteddietfor10daysduringwhichtheyreceivedseven
toninefoodpelletsperdaytomaintaintheirbodyweightat~85%oftheirfreelyfeeding
weight.Suchadietregimewasmaintainedaswellduringtrainingandtestingdays.Thetask
wasperformedduringthedarkphase.
Odourdetectiontestinmice
InordertotesttheabilityofwildtypemicetodetectthesmellofLL,wesubsequentlyintro-
ducedpeppermint(40μloffreshlypreparedwater(distilled)extractfrompeppermintleaves),
LL(40μloffreshlypreparedsolutionindistilledwater,1M,pH=7.4,L1750Sigma,(cid:21)98%
purity),andurine(collectedatthedayoftestingfromfemalemicewithwhichthetestedmale
micehadneverbeenincontact,10μl).TheodoursequenceintroducedtoOlfr78nullmiceand
controlswaswater(40μl),LL(80μloffreshlypreparedsolutionindistilledwater,2M,pH~7.4,
71718Sigma,(cid:21)99%purity),urine(collectedatthedayoftestingfromfemalemicewithwhich
thetestedmicehadneverbeenincontact,10μl),vanillaextract(40μlof1:10solutiondiluted
indistilledwater),distilledwater(40μl).2MLLwaschosenforfurthertestinginOlfr78null
miceandtheirlittermatessince2MLLinducedsimilarbehaviourresponsesincontrolmice
onamixedbackgroundincomparisontoresponsesevokedby1MLLinC57Bl6/Nmice.30
minsbeforetesting,themalemousewasplacedintoafreshtestcage,andthenanodourwas
introducedonapieceofWattmanpaper(2.5cmx2.5cm)inacornerofthecagefor3mins
witha1minintervalbetweendifferentodours.Thetimemicespentsniffinganintroduced
odorouspaperwasmeasuredbyanobserverblindtothetestedsubstances.
Odourhabituation-dishabituationtestinmice
ThisolfactiontestwasemployedtoassesstheabilitytodistinguishbetweenanodourofLL
andothersmells,andanabilitytogetusedtoit.30minsbeforetesting,themousewasplaced
intoafreshtestcage.Eachtestcagewasusedonlyonce.Duringthenext10minsadryand
cleancottonstickwasintroducedintothecageinordertodecreaseunspecificsniffingnot
connectedwiththeodour.LL(freshlypreparedsolutionindistilledwater,1M,pH=7.4,
L1750Sigma,(cid:21)98%purity)wassubsequentlytestedwithpeppermint(freshlypreparedsolu-
tionfrompeppermintleaves)andvanilla(dilutedindistilledwater1:100fromvanillaextract),
anddistilledwaterasanegativecontrol.Eachsmellwasintroducedthreetimesfor2mins
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L-Lactateodourperceptioninrodentsandhumans
witha1mininter-trialintervalonacottonstickwhichwasdippedinthesolutionofthetested
substance.Thetimemicespentsniffingdifferentodourswasmeasuredbyaresearcherblind
totestedsubstances.
Odour-rewardassociativelearninginrats
Anodour-rewardassociativelearningtaskinratswasemployedtoidentifywhetherratsare
abletousethesmellofLLtofindareward.Thistestwasperformedaccordingto[24]with
modifications.
Analuminiumoperantbox(80cmx80cmx50cm)wasused.Theinterioroftheboxwas
spray-paintedmattblacktoavoidthepresenceofvisualcuesandtoprovideenoughcontrast
forthemovement-trackingsoftware,Biobserve(Viewer2version2.2.0.91,BIOBSERVE
GmbH,Bonn,Germany).Spongeswereallidentical,measuring8.5cmx7cmx4cm,witha
2cmholecutinthecentre,andwereonlyusedforonetestingdaywithanindividualanimal.
Kellogg’sFrootLoopsusedinthestudywereshowntobeagoodreinforcementinoperant
tasks[25].Allthesessionswererecordedbyavideocamerapositioneddirectlyabovetheoper-
antboxtoallowobservationduringtrialsandsubsequentoff-lineanalysis.
Fortheexperiments,onespongewasimpregnatedwitheithertestodourofLL(freshlypre-
paredsolutionindistilledwater,1M,pH=7.4,L1750Sigma,(cid:21)98%purity)orcontrolodour
ofalmond(1:100dilutedalmondextractindistilledwater)justbeforeeachtrial.Almondwas
chosenforpositivecontrol,asitisknownthatratscansmellitandseemtohavenopreference
noraversiontoit[26].Thespongewithconcealedrewardwasplacedinadifferentcornerof
theoperantboxforeachtrialaccordingtoapredeterminedsequenceofnumbersfromaran-
domnumbergenerator(1,2or3).Theratwasalwaysplacedinthesamecorner(0)ofthe
box,nosefacingthewallatthestartoftrial,andtheremainingtwocornerswereoccupiedby
emptyspongesimpregnatedwithdistilledwater.Intotal12ratswereusedforconditioning
training,andeachratwastestedwithbothodours.Halfoftheanimalswerefirstconditioned
withalmondodourand,followinga5-daybreak,withLLodour.Theotherhalfwerecondi-
tionedinthereverseorder.
Duringthefirsttrainingday,ratswereallowedtoexplorethearenafor5minbeforeasingle
FrootLoopwasplacedontheflooroftheboxtofamiliarizetheratswiththereinforcement
andtheexperimentalarena.Ratswerelefttoconsumethefoodandthenreturnedtotheir
homecages,andtheprocedurewasrepeated(i.e.twotrainingepisodesforeachrat).During
thesecondtrainingday,asimilarprotocolwasfollowedwiththeexceptionthatthree(odour-
less)spongeswereplacedinthecornersofthebox,andduringthesecondtrialtheFrootLoop
wasplacedintotheholeofaspongetoensurethattheratswereabletoretrievetherewardin
thecourseoftheirnormalforagingbehaviour.
OnthetestingdayafewFrootLoopswerecrushedtoapowderandsprinkledoverthefloor
ofthewholeboxtopreventtheratsfrombeingguidedbythesmellofthehiddenFrootLoop
duringthefollowingtrials.Eachsessionconsistedof6trials.Forthefirsttrialonly(introduc-
torytrial0),thefoodrewardwasplacedonthetopofthespongesothattheanimalscould
visuallylocateitandthistrialwasnotincludedintheanalysis.Thiswasnecessarybecausein
thepreliminaryexperimentswefoundthatwhencrushedFrootLoopswerespreadaroundthe
arena,ratsessentiallyneverfoundaconcealedrewardwithina5minuteperiod.Inthenexttri-
alsthelatencyforacorrectresponse(timetakenforarattoretrievereward)anderrorswere
recorded.Errorswerecountedasanose-pokeinawater-impregnatedsponge,orsniffingat
thecorrectspongewiththeodourwithoutanose-pokeafterwards.Thesamespongeswere
usedforrepeattrialswitheachratbutthepositionofthetargetspongewaschangedrandomly
aftereverytrial.Afterfindingthefoodreward,ratswereallowedtoeatitbeforebeingremoved
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L-Lactateodourperceptioninrodentsandhumans
Table1. Keyoutcomesofthehumanodourdetectiontest.
fractionofparticipantscharacterisedthesmellas*
testsubstance ageofparticipants,years participantsdetectingthesmell pleasant unpleasant neutral
L-Lactate 20.1±0.4 92.6%(87/94) 36.8%(32/87) 19.5%(17/87) 43.7%(38/87)
acetate 20.5±0.6 95.3%(61/64) 6.6%(4/61) 49.2%(30/61) 44.3%(27/61)
propionate 19.9±0.4 98.4%(60/61) 1.7%(1/60) 71.7%(43/60) 26.7%(16/60)
pyruvate 20.0±0.4 96.8%(61/63) 52.5%(32/61) 24.6%(15/61) 23.0%(14/61)
water 20.1±0.4 9.6%(9/94) 11.1%(1/9) 44.4%(4/9) 44.4%(4/9)
*Percentageofparticipantswhoratedthepresentedodoursaspleasant,unpleasantorneutralwascalculatedfromthetotalnumberoftestsubjectswho
detectedanodourinthejar.
https://doi.org/10.1371/journal.pone.0178478.t001
fromtheboxandreturnedtotheirhomecage.Acut-offtimeof5minsand2–5minsofinter-
trialintervalweremaintained.
Odourdetectiontestinhumans
TheprotocolforthisstudywasapprovedbytheUniversityofBristolEthicsCommittee
(approval47121).Averbalconsenttobepartofthestudywasobtainedfromeveryparticipant.
InordertotestwhetherhumanscandetectanodourofLL,werecruited94youngfemale
andmalevolunteers(Table1),andaskedthemtoindicatethepresenceofanodourinfour
jarscontainingvariouschemicalsorwater.Thetestwasperformedduringthreeindependent
dayswithgroupsofmixedgender.Theorderoftheodourpresentationwasrandom,andthe
participantswerenotinformedaboutwhatkindofodourstoexpect.100mlof1M(pH=7.4)
freshlypreparedsolutionsindistilledwaterofLL(L7022Sigma,~98%purity),pyruvate
(P2256Sigma,(cid:21)99%purity),propionate(P1880Sigma,(cid:21)99%purity)oracetate(S2889
Sigma,(cid:21)99%purity)wereputinwideodourlessglassjars,whichwerekeptclosedbetween
tests.Distilledwaterservedascontrol.Theparticipantsweregivenaquestionnairetodeter-
mine(1)whethertheycoulddetectanysmellinthejar(yes/no),(2)iftheyperceivedthissmell
aspleasant,unpleasant,orneutral,and(3)whethertheyperceivedthesmellassweet,sour/
acid,bitter,neutral,or“hardtodefine”.Participantswereinstructedonhowtoperformthe
testandthenexecuteditthemselves.Onlyoneparticipantwaspresentinthetestroomata
time.
Statisticalanalysis
Resultsareexpressedasmean±SEM.Statisticalanalysiswasperformedbyone-ortwo-way
ANOVA,orrepeatedmeasurementsANOVAwithBonferroni’scorrectionasapost-hoctest
formultiplecomparisons,t-test,orFisher’sexacttestcalculatedfromacontingencytable
(PRISM,GraphPad,SanDiego,CA).Toanalysereward-associatedlearninginratsallthe
errordataweretransformedintocorrect/incorrectbinomialdataandevaluatedbycontin-
gencyanalysistocomparedistributions(eitherchi-squaretestfortrendorchi-squaretestfor
goodnessoffit).P<0.05wasconsideredsignificant.
Results
MiceareabletodetectanddiscriminatetheodourofL-Lactate
First,wetestedinanodourdetectiontaskwhethermiceareabletosmellLL.Micespentsignif-
icantlymoretimesniffingLL-impregnatedpapercomparedtowater(LL13.59±1.84secvs
water4.84±0.64sec,p<0.01),whichwasclosetotheamountoftimetheyspentsniffing
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L-Lactateodourperceptioninrodentsandhumans
Fig1.MiceareabletodetectanddiscriminatetheodourofL-Lactate.A.Odourdetectiontask.Mice
spentsignificantlymoretimesniffingL-Lactate(1M)thanwater,andthesameamountoftimeaspeppermint
extract.Dataareshownasmean±SEM,n=8.*p<0.05,**p<0.01,***p<0.001vswater,pairedt-test.B.
Odourhabituation-dishabituationtest.Onthefirstpresentation,micespentsignificantlymoretimesniffing
L-Lactateincomparisontowaterandtothethirdintroductionofthepreviousodour(peppermint).Over
severalintroductionsmicehabituatedtothesmellofL-Lactateandsniffeditforshortertimesduringthethird
introductioncomparedtothefirstDataareshownasmean±SEM,n=3.#p<0.05vsthirdintroductionof
previousodour,*p<0.05vsfirstwaterintroduction,pairedt-test.C.OdourdetectiontaskinOlfr78null
mice.Olfr78nullmicespentthesameamountoftimeascontrollittermatessniffingL-Lactate(2M),orcontrol
smells–vanillaandurineoftheoppositegender.Dataareshownasmean±SEM,n =7,n =9.
Olfr78null WT
https://doi.org/10.1371/journal.pone.0178478.g001
peppermint(LL13.59±1.84secvspeppermint10.68±1.79sec,p>0.05,Fig1A).Micespent11
timeslongersniffingapapercontainingurineincomparisontowater(urine52.12±3.85secvs
water4.84±0.64sec,p<0.001,Fig1A),and4timeslongerincomparisontoLL(urine52.12
±3.85secvsLL13.59±1.84sec,p<0.001,Fig1A).Thisisnotsurprisingbecauseforrodents
urineoftheoppositegenderisoneofthemostpotentodorantsignals.
Inanotherbehaviourparadigm,weintroducedeacholfactorystimulusthreetimes(water,
peppermint,vanilla,andLL;Fig1B).Micehadlittleinterestinwaterbutwhenanyofthe
odourswasintroducedforthefirsttime,theyspentasignificantlengthoftimesniffingthem
(sniffingtimes:11.6±1.82secforpeppermint1,13.17±2.15secforLL1,and14.27±0.78secfor
vanilla1vs3±1.02secforwater1,p<0.05).Whenanyoftheodorantswasintroducedrepeat-
edly,sniffingtimesreducedfromthefirsttothethirdpresentation.Forexample,forLLdiffer-
enceswereasfollows:secondvsfirstpresentation:1.63±1.17secvs13.17±2.15sec,p<0.01;
thirdvsfirstpresentation:3.33±0.48secvs13.17±2.15sec,p<0.05.Micecoulddifferentiate
theodourofLLfromthepreviouslyintroducedpeppermint(2.1±0.15secvs13.17±2.15sec,
peppermint3vsLL1,p<0.05).ThesmellofLLwasalsodifferentiatedfromthesubsequently
introducedvanillaasthesniffingtimeincreasedagain(lastpresentationofLL3.33±0.48secvs
firstvanillapresentation14.27±0.78sec,p<0.01).
Olfr78nullmicewerenotdifferenttowildtypecontrolsintheirabilitytodetecttheodour
ofLL,andspentsimilaramountoftimesniffingodoursofLL,vanillaorurine(Fig1C).Partic-
ularly,Olfr78nullmicespentsignificantlymoretimesniffingLL-,vanilla-orurine-impreg-
natedpapercomparedtowater(water3.87±0.68secvsLL6.67±0.83sec,p<0.05;vanilla9.62
±1.67sec,p<0.01;urine32.56±3.2sec,p<0.0001).
RatscanusetheodourofL-Lactateasacue
TotesttheabilityofratstodetecttheodourofLLweemployedanodour-rewardassociative
learningtask(Fig2A).DuringthefirsttestinvolvingahiddenFrootLoop(trial1),ratstooka
similaramountoftimetofindtherewardusingthesmellofLLoralmond(21.08±4.36secvs
26.98±7.78sec,LLvsalmond,p>0.05,Fig2B).Thefollowingtrials(2–5)revealedtheeffectof
trialnumberonthelatencytofindtherewardbysmell(F(4,44)=4.610,P<0.01,Fig2B),
whilethechoiceoftheodorantmadenosignificantdifference(F(1,11)=0.06604,P>0.05,
Fig2B).Duringthelasttrial(5),ratsfoundtherewardmuchquickerthaninthebeginning
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L-Lactateodourperceptioninrodentsandhumans
Fig2.RatsareabletodiscriminatetheodourofL-Lactate.A.Schematicillustrationofanodour-
rewardassociativelearningtaskinrats.Duringthetrainingdays,ratswerehabituatedtothearenaand
rewardretrievalprocedure.Duringtheintroductorytrial(0)onthetestingday(notincludedinfurtheranalysis),
aspongesoakedwiththetestodorantcarriedavisiblefoodreward.Duringconsequentfivetrialsonthe
testingday,arandomlyplacedspongesoakedwiththetestsmelldispensedahiddenfoodreward.
Outcomesofodourassociativelearningtaskinrats:B.Timetakenbyratstofindafoodrewardbythe
smell(latency)wasnotdifferentbetweenL-Lactateandapositivecontrol,almond.Overthetrialsthelatency
tofindafoodrewarddecreasedforbothodorants.Dataareshownasmean±SEM,n=12.*p<0.05,
**p<0.01,two-wayANOVAwithBonferroni’scorrection.C.Distributionandmeanofcorrecttrialsforratsdid
notdifferbetweenL-Lactateandalmond.Numberofobservedcorrecttrialswassignificantlyhigherthan
expectedbychance.Dataareshownasmean±SEM,n=12.###p<0.001,##p<0.01vsmeanexpectedby
chance,t-test.
https://doi.org/10.1371/journal.pone.0178478.g002
(trial1vstrial5,p<0.5,Fig2B),irrespectiveoftheodorantcue.Next,weassessedhowmany
ratssucceededtofindarewardwithoutanyincorrectnosepokes.LLwasequaltoalmondin
thisregard(p>0.05,Fig2C),andtheprobabilityofacorrectchoicewashigherthanwhatmay
beexpectedbychance(p<0.001foralmond,p<0.01forLL,Fig2C).Theprobabilityofran-
domlychosencorrectresponseswascalculatedfromtheassumptionthatiftheratsdonotdis-
tinguishanodourofeitherLLoralmond,theywouldhave1/3ofchancepertrialforacorrect
nosespokebetweenthreesponges.Whencalculatedover5trials,thenumberofrandomly
chosencorrectchoiceswouldbe1.67(Fig2C).Thedistributionofchancewasmodelled
usingbinomialtheoremtocalculateexpectedproportionofratsforeachnumberofcorrect
responses.Additionally,achi-squareforgoodnessoffittestedforadiscrepancybetween
theobserveddistributionsforalmondandLLandtheexpecteddistributionbychanceand
returnedverylowp-valuesforboth(p<0.0001forbothodourgroups).Altogether,thesedata
indicatethattheodourconditioningenabledratstocorrectlyidentifylocationofthereward
usingbothLLoralmondodoursasolfactorycues.
HumansareabletodetecttheodourofL-Lactate
YoungfemaleandmalevolunteerswereaskedtosmelljarscontainingsolutionsofLL,acetate
orpropionatewhichwerereportedtoalsoactivateOlfr78invitro[18,20].Pyruvatewas
includedasamoleculecarryingthesamecaloricvalueasLLandchemicallysimilartoLL,but
whichwasnotpreviouslydescribedasanolfactorystimulant,noraligandforOlfr78.92.6%of
theparticipantsindicatedthepresenceofasmellinajarcontainingasolutionofLL,36.8%
and19.5%outofwhichcharacterisedthissmellaspleasantandunpleasant,respectively
(Table1).Interestingly,96.8%ofthevolunteersdetectedanodorantinajarcontainingpyru-
vate,againmainlycharacterisingitssmellaspleasant(52.5%vs24.6%,Table1).Tonote,the
proportionofparticipantsindicatingpyruvateasapleasantodourwashighercomparedtoLL
(pyruvate52.5%vs.LL36.8%,p<0.0001).Acetateandpropionatewerecharacterisedas
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L-Lactateodourperceptioninrodentsandhumans
Fig3.Gender-dependentdifferencesinL-Lactateodourperceptionbyhumans.A.Gender-
dependentdifferencesinodourdiscrimination.Similarpercentagesoffemaleandmaleparticipantswere
abletodiscriminateanodourinsolutionsofL-Lactate,pyruvate,acetateandpropionate.Numbersindicate
amountofparticipantstestedwithasmell.B.Gender-dependentdifferencesin‘unpleasant’odour
perception.L-Lactateandpyruvatewereperceivedasrepulsivebymorewomenthanmen(L-Lactate:
25.9%vs6.9%;pyruvate:33.3%and9.1%).Similarpercentagesoffemaleandmaleparticipantsratedthe
odoursofacetateandpropionateasunpleasant.C.Gender-dependentdifferencesin‘pleasant’odour
perception.Almosttwicemorementhanwomenratedthesmellofpyruvateasappealing(72.7%vs41%).
SimilarpercentagesofmenandwomenratedtheodourofL-Lactate,acetateandpropionateaspleasant.D.
Gender-dependentcharacterisationoftestodours.L-Lactate,pyruvate,acetateandpropionatewere
ratedsimilarbymenandwomeninrespecttothedimensionoftheirsmell/taste.Mostparticipants,
independentofgender,indicatedthemassweetandsour/acid,respectively.Lactateandwater:n(F)=64,n
(M)=30;acetate:n(F)=47,n(M)=17;propionate:n(F)=41,n(M)=21;pyruvate:n(F)=40,n(M)=23.
*p<0.05,Fisher’sexacttest.
https://doi.org/10.1371/journal.pone.0178478.g003
odoroussubstancesby95.3%and98.4%,respectively,withalargemajoritycharacterising
themasunpleasant(Table1).
Therewasnosignificantdifferencebetweenmenandwomeninthefrequencyofdetection
ofanyoftheodours(Fig3A).However,therewasadifferenceinhowLLodourwasperceived
bymenandwomen,sincemanymorewomencharacteriseditasunpleasant(25.9%vs6.9%,
p<0.05)(Fig3B).Curiously,menalsodidnotfindthesmellofpyruvateasunpleasantas
womendid(33.3%vs9.1%,p<0.05).ThefrequencyofreportingLLasapleasantsmellwas
notgenderdependentbutforpyruvatetheproportionofmenlikingitwassignificantlygreater
(72.7%vs41%,p<0.05,Fig3C).Irrespectiveofgender,odoursofacetateandpropionatewere
perceivedasanunpleasantbymostoftheparticipants(Fig3B).
Perceptionofsmellishighlysubjective.Whenparticipantswereaskedtocharacterisetheir
perceptionoftheodorants,LLandpyruvatewereoftendescribedasasweetsubstance,while
acetateandpropionateweremostlycharacterisedassour/acid(Fig3D).However,morepartic-
ipantsindicatedpyruvateodourassweetincomparisontoLL(LL27.6%vs52.5%pyruvate
p<0.01).Onlyafewparticipants(9outof94)indicatedapresenceofodourinajarcontaining
waterwhichapproximatestherateoffalsedetectioninthistest(p<0.0001forwatervsany
othertestedodour,Fisher’sexacttest,Table1).
Discussion
LLisaremarkablemoleculewhichstandsoutfromthemyriadofchemicalswhichsurround
usineverydaylife,becauseitispivotaltometabolismofallcellsandispresentinfoodsand
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L-Lactateodourperceptioninrodentsandhumans
bodysecretions[27–30].AfterthediscoveryofLL-sensitiveGPCRsinthebrain[6,7,11],
LLwasrecognisedasapotentialsignallingmolecule,raisingtheinterestinanypossiblerecep-
torsforthismolecule.Inhumans,ORsconstitutehalfoftheknownGPCRs(http://www.
guidetopharmacology.org),andrepresentabiggroupofpotentiallyLL-sensitiveGPCRssome
ofwhicharefoundoutsideoftheolfactoryepitheliumandprobablyimportantirrespectiveof
theirroleinolfaction.Here,wetestedwhetherLLcanserveasanodorantinrodentsand
humans,andassessedpotentialcontributionofOlfr78inLLodourperception.Tothisend,we
performedseveralodourdetectionorodour-rewardassociativelearningtasksinrodentsand
humans.InordertodelineatetheinvolvementofOlfr78insensingLL,weusedanOlfr78null
mousemodelinanodourdetectiontask.
HereforthefirsttimeweshowthatmiceareabletodetectthesmellofLLanddiscriminate
itfrompeppermintandvanillaodours.Ratswereabletoperformanodour-rewardassociative
learningtaskusingthesmellofLLasacue,whichworkedapproximatelyaswellasastandard
odorant,almond.
Whentestedinanodourdetectiontask,Olfr78nullmicehadthesamebehavioural
responsesascontrolmicetothesmellofLL.Thisisnotentirelysurprisingbecausethereare
onlyveryfewcaseswhenanablationof(oramutationin)oneoftheORswouldleadtoa
changeinodour-evokedbehaviour.OneofsuchORsisodr-10inC.elegans,ablationofwhich
leadstothelossofchemotaxisspecificallytodiacetyl,butnottoothersubstancesknownto
causechemotaxisinthisspecies[31].Twofurtherexamplesofsuchreceptorsinmiceare
trace-amineassociatedreceptor(TAAR)4and5,ablationofwhichresultedinlackofaversion
topredatorurine[32]orlackofattractiontomurineurine[33],respectively.
ThereisapossibilitythatthesensitivitytoLLwasnotcompletelylostbutonlyreducedin
Olfr78nullmice.TestingresponsestolowerLLconcentrationsinodourdiscriminationtask
mighthelptofurtherclarifythisissue.
Ontheotherhand,odourdetectionandcodingistypicallycombinatorial.Thesameodour
isusuallydetectedbymorethanonereceptorwhilethesamereceptormayberesponsiblefor
thedetectionofdifferentodours[34,35].Basedonthesequencesimilarity,Olfr558(human
orthologueOR51E1)isassignedtothesamesubfamilyasOlfr78anditisknownthatORsof
thesamefamilyfrequentlydetectodorantswithsimilarstructures[35,36].Indeed,itwas
shownthatOlfr558isactivatedbypropionate[37,38].ItisthereforepossiblethatOlfr558
mightbeabletointeractwithLLandcouldexplainwhyOlfr78deletioninmicedidnotpre-
ventdetectionofLL.Interestingly,Olfr558isalsoexpressedoutsideoftheolfactorysystem
[20].
TwootherORs,OR1G1andOR52D1,wererecentlyshowntobesensitivetopropionate
[37,39],buttheyhavenotyetbeentestedfortheabilitytosenseLL.BLASTsearchusingthe
cDNAsequenceofOlr59(ratanalogueofOlfr78,GenBankAY317512.1)asatagrevealed
multipleolfactoryreceptorgeneswithmorethan60%similaritytoOlfr78.Onesuchageneis
Olfr406,whichis96%identicaltoOlfr59attheDNAlevel.Thereisnoinformationonthe
ligandsofthisreceptorbutitmightbeyetanotherORresponsibleforLLdetectioninOlfr78
nullmice.
Whentestedinhumans,lessthan10%ofparticipantswereunabletodetecttheodourof
LL.Interestingly,besidesLL,thechemicallyrelatedcompoundspropionateandacetatewere
reportedtoactivatethehumanorthologueofOlfr78.However,subjectivelythesesubstances
wereperceiveddifferentlybytheparticipants.Importantly,allchemicalswereneutralisedand
theeffectsofprotonswhichcouldberesponsibleforsomeaspectsofsmellperceptionwas
thereforeminimal.LLwasusuallyqualifiedasaratherpleasantodourwithasweetscent,and
pyruvatewhichhasnotbeentestedasapotentialligandoftheORsdescribedabove,hadasim-
ilarodourprofileasLL.ItislikelythathumanscandistinguishbetweenLLfrompyruvate
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