Ultrasensitive Detection of Amines by a Trace Amine-Associated Receptor

12 Pages·2013·4.03 MB·English

3228•TheJournalofNeuroscience,February13,2013•33(7):3228–3239
Cellular/Molecular
Ultrasensitive Detection of Amines by a Trace
Amine-Associated Receptor
JingjiZhang,1*RodrigoPacifico,1*DillonCawley,1PaulFeinstein,2andThomasBozza1
1DepartmentofNeurobiology,NorthwesternUniversity,Evanston,Illinois60208,and2DepartmentofBiologicalSciences,HunterCollege,CityUniversity
ofNewYork,NewYork,NewYork10065
ThemammalianmainolfactorypathwaydetectsvolatilechemicalsusingtwofamiliesofG-protein-coupledreceptors:alargerepertoire
ofcanonicalodorantreceptorsandamuchsmallersetoftraceamine-associatedreceptors(TAARs).TheTAARsareevolutionarily
conservedinvertebrates,includinghumans,suggestinganindispensibleroleinolfaction.However,littleisknownaboutthefunctional
propertiesofTAARswhenexpressedinnativeolfactorysensoryneurons.Herewedescribeexperimentsusinggenetargeting,electro-
physiology,andopticalimagingtostudytheresponsepropertiesofTAAR-expressingsensoryneuronsandtheirassociatedglomeruliin
mice.WeshowthatolfactorysensoryneuronsthatexpressasubsetoftheTAARrepertoirearepreferentiallyresponsivetoamines.In
addition,neuronsexpressingspecificTAARs,TAAR3orTAAR4,arehighlysensitiveandarealsobroadlytuned—respondingtostruc-
turallydiverseamines.Surprisingly,wefindthatTAAR4isexquisitelysensitive,withapparentaffinitiesforapreferredligand,phenyl-
ethylamine,rivalingthoseseenwithmammalianpheromonereceptors.Weprovideevidencethatthisunprecedentedsensitivityis
mediatedviareceptorcouplingtothecanonicalodoranttransductioncascade.ThedatasuggestthattheTAARsareevolutionarily
retainedintheolfactoryreceptorrepertoiretomediatehigh-sensitivitydetectionofabiologicallyrelevantclassofodorousstimuli.
Introduction and these receptors have been shown to be narrowly tuned to
Todetectstructurallydiversevolatilechemicals,themammalian amines(LiberlesandBuck,2006;Ferreroetal.,2011;Ferreroet
olfactorysystemhasdevelopedalargerepertoireofchemosen- al.,2012).IthasbeensuggestedthatidentifiedTAARligandsmay
sory receptor genes. There are two known families of seven- serve as social cues and/or predator-derived chemicals (kairo-
transmembrane receptors expressed by olfactory sensory mones)inrodents.Inparticular,trimethylamineisenrichedin
neurons(OSNs)inthemainolfactoryepitheliumofthemouse: the urine of sexually mature male mice and activates TAAR5
(cid:1)1000canonicalodorantreceptors(ORs)andamuchsmaller (Liberles and Buck, 2006). (cid:1)-Phenylethylamine is enriched in
familyoftraceamine-associatedreceptors(TAARs)(Liberlesand carnivore urine, elicits aversive responses in rodents, and acti-
Buck,2006;Neietal.,2008).AlthoughbothORsandTAARsare vates TAAR4 (Ferrero et al., 2011). The data suggest that the
G-protein-coupledreceptors,theTAARsaremorecloselyrelated TAARsmaybespecializedtodetectchemicalsignalsthatcarry
tobiogenicaminereceptors(Lindemannetal.,2005;Liberlesand specificinformationorthathaveanegativevalence.
Buck,2006)andareevolutionarilyconservedinvertebrates,in- Recently, it was shown that TAAR-expressing OSNs form a
cluding humans (Lindemann et al., 2005; Hashiguchi and distinctprojectiontoglomeruliinthedorsalaspectofthemain
Nishida,2007;Hussainetal.,2009),suggestingthattheyservea olfactorybulb(Johnsonetal.,2012;Pacificoetal.,2012).More
criticalchemosensoryfunction. specifically,10ofthe14TAARgenesareselectivelyexpressedby
Inthemouse,14ofthe15intactTAARgenesareexpressedin a subset of OSNs and are mapped to clustered glomeruli that
theolfactorysystem(LiberlesandBuck,2006).Ligandshavebeen respondselectivelytolowconcentrationsofamines(Pacificoet
identifiedforsixmouseTAARsusingheterologousexpression, al.,2012).However,therearecurrentlynodataexaminingthe
odorant response properties of OSNs that express identified
TAARgenesinanyspecies.Consequently,littleisknownabout
ReceivedSept.8,2012;revisedNov.19,2012;acceptedDec.13,2012.
the functional specificity of TAARs in vivo or whether TAAR
Authorcontributions:R.P.,P.F.,andT.B.designedresearch;J.Z.,R.P.,D.C.,andT.B.performedresearch;J.Z.,R.P.,
ligands identified in vitro activate their corresponding TAAR-
andT.B.analyzeddata;R.P.andT.B.wrotethepaper.
*J.Z.andR.P.contributedequallytothiswork. expressingOSNs.Inaddition,thetransductioncascadethatme-
ThisworkwassupportedbyNationalInstitutesofHealth/NationalInstituteonDeafnessandOtherCommunica- diatesTAARresponsesintheirnativeneuronsisunknown.
tionDisordersGrant1R01DC009640(T.B.),theWhitehallFoundation(T.B.),andBrainResearchFoundation(T.B.),
Weusedgenetargeting,electrophysiology,andinvivoimag-
andNationalInstitutesofHealth/NationalInstituteofGeneralMedicalScienceGrant5SC1GM088114(P.F.).We
ingtocharacterizethefunctionalpropertiesofTAAR-expressing
thankAdamDewanandMattWachowiakforcommentsonthemanuscriptandtheGeneTargetingandTransgenic
ResourceCentersatRockefellerUniversityforassistancewithgenetargeting. OSNsandtheircorrespondingglomeruliinmice.Ourdatashow
Theauthorsdeclarenocompetingfinancialinterests. thatTAAR-expressingOSNsareselectively(althoughnotexclu-
CorrespondenceshouldbeaddressedtoThomasBozza,DepartmentofNeurobiology,NorthwesternUniversity, sively) responsive to amines. OSNs expressing TAAR3 and
2205TechDrive,Hogan2-160,Evanston,IL60208.E-mail:[email protected].
TAAR4 and their corresponding glomeruli are more broadly
DOI:10.1523/JNEUROSCI.4299-12.2013
Copyright©2013theauthors 0270-6474/13/333228-12$15.00/0 tunedthanexpected,andTAAR4isextraordinarilysensitive,ex-
Zhang,Pacificoetal.•UltrasensitiveAmineDetection J.Neurosci.,February13,2013•33(7):3228–3239•3229
hibitingrobustresponsestosubpicomolarodorantconcentra- daverine, cyclohexylamine, propylamine); aldehydes (butyraldehyde,
tions, comparable with mammalian pheromone receptors benzaldehyde,heptaldehyde,octylaldehyde);acids(isovalericacid,pro-
(Leinders-Zufall et al., 2000). We provide evidence that the pionicacid,butyricacid,hexanoicacid);orketones(isovalerophenone,
TAARresponsesarelikelymediatedbycouplingtothecanonical acetophenone,butyrophenone,propiophenone).
AnalysisandcurvefittingwereperformedusingIgorPro4.08J(Wave
odoranttransductioncascade.Together,ourdataarguethatthe
Metrics). Dose–response curves were fitted by the Hill equation: I (cid:2)
TAARs, and TAAR4 in particular, serve as high-affinity amine
I /(1(cid:7)(EC /C)n),whereIrepresentsthepeakresponse,I themax-
detectorsinmammalianolfactorysystems. max 50 max
imumcurrentinducedbythesaturatingconcentration,EC theconcen-
50
tration at which the half-maximum response was reached, C the
MaterialsandMethods concentrationofodorant,andntheHillcoefficient.Statisticaltestswere
AllprocedureswereapprovedbytheNorthwesternUniversityInstitu- performedusingStatview.Thettestwasusedformeasuringthesignifi-
tionalAnimalCareandUseCommittee. cance of difference between two distributions. All data are shown as
Gene targeting. Generation of the Venus3Taar4, Taar4–IRES– mean(cid:5)SEM.
tauCherryandTaar3–IRES–tauVenustargetedstrainswasdescribedpre- Histology.Whole-mountimagesofolfactoryepitheliaandbulbswere
viously(Pacificoetal.,2012).TogeneratethehumanTAAR5(hTAAR5) performed using confocal microscopy on fresh, unfixed tissue. Com-
swap(hTaar53Taar4–IRES–tauVenus),thecodingsequenceofhTaar5 binedimmunohistochemistry/insituhybridizationwasperformedasde-
was amplified from a bacterial artificial chromosome (BAC) clone scribed previously (Ishii et al., 2004; Bozza et al., 2009) using
(RP11–295F4), flanked with AscI restriction sites, and preceded by a homozygousT4–ChR–YFPmice(Pacificoetal.,2012).Inshort,P21–
Kozakconsensussequence.TheTAAR4targetingvectorwasmodifiedso P30micewereanesthetizedandfixedthroughcardiacperfusionwith
thatthenativecodingsequencewasreplacedwithAscIandPacIsites. ice-coldheparinizedsalineand4%paraformaldehyde.Nasaltissuewas
ThehT5codingsequencewasinsertedintotheAscIsite,followedbya dissectedandpostfixedat4°Covernight,followedby0.5MEDTAdecal-
PacIcassettecontainingtheself-excisingneogeneACNf(Buntingetal., cificationand30%sucrosecryoprotection(both4°Covernight).OCT-
1999),aninternalribosomeentrysite,andthecodingsequencefortau- embedded epithelium was frozen and sectioned at 12 (cid:2)m. YFP was
Venus, a fusion between the microtubule-associated protein tau and detectedusingarabbitpolyclonalantibodytoAequoreavictoriagreen
Venusyellowfluorescentprotein(YFP)(Nagaietal.,2002).Thetarget- fluorescentprotein(GFP)(Clontech).Riboprobestosignaltransduction
ingvectorwaslinearizedwithXhoIandelectroporatedintoE14EScells components were generated via in vitro transcription from PCR-
usingstandardmethods.G418-resistantcloneswerescreenedbySouth- amplified templates with an incorporated T7 RNA polymerase pro-
ernblotusingprobesexternaltothetargetingvector.Chimeraswere moter. Gene fragments were amplified from BAC DNA or from an
generatedbyblastocystinjectionintoC57BL/6embryos. olfactoryepithelialcDNAlibrary.Probesequencesareavailableonre-
Electrophysiological recordings. Patch-clamp recordings were made quest. All imaging was performed using a Carl Zeiss LSM5 confocal
fromthedendriticknobsofsemi-intactOSNsinepithelialexplants(Ma microscopeandanalyzedwithCarlZeissAIMsoftware.
etal.,1999).Maleandfemalemice(n(cid:2)52;P7–P20)wereanesthetized Invivoimaging.Glomerularimagingwasdoneasdescribedpreviously
withketamine–xylazine(200and15mg/kg,i.p.,respectively),andthe (Pacificoetal.,2012)withsomemodifications.Maleandfemalemice
olfactoryepitheliumwasremovedandkeptinoxygenatedbathsolution were 8–12 weeks old and were heterozygous for both the OMP–spH
(95%O2–5%CO2)containingthefollowing(inmM):124NaCl,3KCl, (Bozzaetal.,2004)aswellasT4–RFPand/orT3–YFPalleles(Pacificoet
1.3MgSO ,2CaCl ,26NaHCO ,1.25NaHPO ,and15glucose,pH7.4 al.,2012).Micewereanesthetizedwithurethane(1g/kg,i.p.;Sigma)and
4 2 3 4
(305mOsm).Theepitheliumwastransferredtoarecordingchamber chlorprothixenehydrochloride(10mg/kg),followedbyadministration
andkeptundercontinuousflow(1–2ml/min)ofoxygenatedbathsolu- ofatropinesulfate(2.7mg/kg;Med-Pharmex).Micewerefreelybreath-
tion.Allexperimentswereperformedatroomtemperature. ing throughout the experiment. For some experiments, imaging was
The knobs of OSNs were visualized with an upright epifluores- donefromonesidewhileoccludingthecontralateralnaris.
cence/infrared differential interference contrast (DIC) microscope Odorantswereappliedusingacustom-made,flowdilutionolfactom-
equippedwithacooledCCDcamera(SensiCamQE;CookeCorpo- eterandcontroller(LASOM;RPMetrix)thatwasdesignedtoavoideven
ration) and a 40(cid:3) water-immersion objective. Dendritic knobs of minutelevelsofcross-contaminationamongtheamines,whichcouldbe
labeled OSNs were identified under fluorescence illumination and easily detected by the most sensitive TAAR glomeruli. To obtain low
targetedunderDIC.Patchpipetteswerepulledfromborosilicateglass enoughconcentrations,aminesweredilutedinwaterandsubsequently
withaP-97horizontalpuller(SutterInstruments)andfire-polished byflowdilution.DatawereacquiredusingaNeuroCCD-SM256camera
usingamicroforge(MF83;Narishige).Electrophysiologicalrecord- andNeuroplexsoftware(RedShirtImaging).Blanktrialsweresubtracted
ings were made using an EPC-10 amplifier controlled with Pulse fromodortrialsbeforeanalysistocompensateforphotobleaching.Re-
software(HEKA).Perforatedpatchclampwasperformedbyinclud- sponsemapswereobtainedbysubtractinga3.2stemporalaveragepre-
ing260(cid:2)MamphotericinBintherecordingpipette,whichwasfilled ceding the stimulus from a 3.2 s temporal average encompassing the
withthefollowingsolution(inmM):70KCl,53KOH,30methane- responsepeak.Responsesareexpressedas(cid:8)Ftoaccountforthefactthat
sulfonicacid,5EGTA,10HEPES,and70sucrose,pH7.2withKOH thebackgroundsynaptopHluorin(spH)fluorescenceisnotcorrelated
(310mOsm).Theelectrodeshadtipresistancesrangingfrom8to10 withthepoolofindicatorthatreportsneuronalactivity(Bozzaetal.,
M(cid:4)whenfilledwithinternalsolution.Theliquidjunctionpotentials 2004;McGannetal.,2005).Forrecordingsfromidentifiedglomeruli,
werecorrectedinallexperiments.Signalswereacquiredat10kHzand oneglomeruluswaschosenpermousebecausemultipleglomeruliwithin
low-passfilteredat2.9kHz. thesamemousearenotindependent.Stimuliwerepresentedatleast
Odorantswereappliedwithapressureejectionsystem(PDES-02D; twiceinagivenexperiment.Datashownareaveragesoftwoormore
NPIElectronics)viaamulti-barrelpipetteplaced20(cid:2)mdownstreamof trialsorthefirsttrialincasesinwhichsignificantadaptationofthe
thedendrite.Stimuliweredeliveredusingapulselengthof200ms.No amineresponsewasobserved.Imageswereprocessedandanalyzedin
responseswereseentopressureejectionofsalinevehicleinanycells. Neuroplex(RedShirtImaging)andNIHImageJsoftware.Vaporcon-
Stimulusvolumewasmeasuredbyejectingsalineintomineraloiland centrationswereestimatedusingpublishedvaporpressures(Estima-
measuringthediameteroftheresultingbubbleusingacalibratedeye- tionProgramsInterfaceSuite,version4.0;EnvironmentalProtection
piecemicrometer.Theaverageejectedvolumefora200mspulsewas Agency).
5.8 (cid:5) 1.3 nl (n (cid:2) 20 observations). Amine odorants were dissolved Results
directly in bath solution or were solubilized in dimethylsulfoxide for
TAAR-expressingOSNsareselectivelyresponsivetoamines
some experiments. Identical results were obtained under both condi-
tions.Stockodorantsolutionswerestoredat(cid:6)20°Canddilutedbyadd- ToexaminetheresponseprofilesofTAAR-expressingOSNs,we
ingbathsolution.Odorantmixturescontainedthefollowingodorants: recordedfromgeneticallyidentifiedOSNsingene-targetedmice.
amines((cid:1)-phenylethylamine,trimethylamine,N-methylpiperidine,ca- Odorant-evoked currents were measured by targeted patch-
3230•J.Neurosci.,February13,2013•33(7):3228–3239 Zhang,Pacificoetal.•UltrasensitiveAmineDetection
clamprecordingsinvoltage-clampmodefromfluorescentlyla- (n(cid:2)9cells).At10nM,T3–OSNsrespondedmorespecificallyto
beled dendrites in an epithelial explant preparation (Ma et al., isopentylamine(75.3(cid:5)8.2pA)andcyclohexylamine(47.7(cid:5)7.7
1999;Grosmaitreetal.,2006). pA;n(cid:2)7cells;Fig.2E).Becauseoflimitationsinspaceclamp,OSNs
To gain an overview of the odorant specificity of OSNs ex- oftenexhibitedactionpotentialsridingontopoftherecordedodor-
pressingavarietyofTAARs,wetookadvantageofagene-targeted evokedcurrents(Fig.2D).Interestingly,evensmall-amplitudecur-
mousestrain((cid:8)T4–YFP)inwhichthecodingsequenceforthe rent responses elicited increases in action potential firing rate,
Taar4geneisreplacedwiththatofafluorescentmarker,Venus indicatingthatresponsestoalloftheseaminesarelikelytransmitted
YFP.WeshowedpreviouslythatTAAR-expressingOSNsexhibit tothebulb.Thus,T3–OSNsaremorebroadlytunedthanwouldbe
biasedgenechoiceandarerestrictedtochoosefromamongthe predictedbasedonpreviousinvitroexpressiondata(Liberlesand
TAAR gene repertoire (Pacifico et al., 2012). More precisely, Buck,2006).
OSNsthatchoosetheT4locusfrequentlychoosefromnineother Next, we examined the response profiles of OSNs expressing
dorsallyexpressedTAARgenes.Thus,inheterozygousmice,the TAAR4(T4–OSNs)usingthesamesetofamines.Todothis,we
populationofOSNsexpressingthe(cid:8)T4–YFPallele((cid:8)T4–OSNs) usedtheTaar4–IRES–tauCherrystraininwhichOSNsexpressing
coexpress10ofthe14TAARs(includingtheintactT4allelein theendogenousTaar4(T4)genearelabeledwitharedfluorescent
trans), giving rise to a labeled, diversified population of OSNs protein(RFP)(Fig.3A–C).T4hasbeenreportedtorespondexclu-
(Fig.1A,B).Bysamplingthispopulation,wecouldtherebyfunc- sivelyto(cid:1)-phenylethylamineandstructurallysimilarcompoundsin
tionally characterize sensory neurons that express a variety of vitro (Liberles and Buck, 2006; Ferrero et al., 2011). Like T3, we
TAARgenes.TodeterminewhetherTAAR-expressingOSNsare foundthatT4–OSNswerebroadlytuned,respondingtotheprevi-
selectivelyactivatedbyamines,wetested(cid:8)T4–OSNswithfour ouslyidentifiedligand(cid:1)-phenylethylamine,butalsotootherstruc-
odorantmixtures,eachcomprisingacids,aldehydes,ketones,or turallydiverseamines(Fig.3D,E).Inaddition,weobservedthat
amines(Fig.1B,C).Ahighstimulusconcentrationwasusedto T4–OSNs were strikingly sensitive, exhibiting broad tuning and
uncover even low-sensitivity responses. As a positive control, largeresponsesat10nM(n(cid:2)13cells).Atalowerconcentration(0.5
OSNswerealsotestedwiththephosphodiesteraseinhibitoriso- nM), (cid:1)-phenylethylamine was clearly the preferred stimulus. The
butylmethylxanthine(IBMX),whichiscommonlyusedtoacti- averageamplitudewas112.1(cid:5)19.3pAfor(cid:1)-phenylethylamineand
vatetheodoranttransductioncascade(Firesteinetal.,1991). 8–22pAfortheotheramines(n(cid:2)9cells).Athigherconcentrations
Thevastmajorityof(cid:8)T4–OSNs(44of46OSNs)exhibited (1(cid:2)M),T4–OSNsexhibitedvery-large-amplituderesponsestoall
responsestotheaminemixture.Halfofthecellsrespondedex- thetestedamines(Fig.4C),muchlargerthanwhatisobservedforT3
clusivelytothismixture,whereastheotherhalfrespondedpref- (Fig.2E).Thus,ourrecordingsshowthatbothT3andT4arediffer-
erentiallytotheaminemixtureandexhibitedsmallerresponses entiallysensitivebutalsobroadlytunedtoamines.
tonon-aminemixtures(Fig.1C,D).Forcomparison,wetested
themixturesonOSNsexpressingclassIorclassIIdeletionalleles TAAR4expressionissufficienttoaccountforthebroad
((cid:8)S50–OSNsand(cid:8)M72–OSNs)thatpreferentiallyexpressclass tuningofTAAR4–OSNs
IorclassIIORs,respectively(Bozzaetal.,2009).Neitherpopu- Onepossibleexplanationforthepronouncedbreadthoftuningis
lation showed the same selectivity for the amine mixture (Fig. that TAAR-expressing OSNs could express multiple receptors.
1E). We note that the amine mixture frequently elicited large- TodeterminewhetheralloftheresponsesweobservedinT4–
amplituderesponsesin(cid:8)T4–OSNs—muchlargerthanwhatis OSNsareattributabletotheexpressedT4receptor,wegenetically
typicallyseenwithcanonicalORsatsimilarconcentrationsinthis swappedtheTAAR4codingsequencetoseewhetherthiswould
assay(ourunpublisheddata).Thisobservationindicatedtous eliminateresponsestoalloftheeffectivestimuli.Asimilarap-
thatatleastsomeoftheTAARsmightbeverysensitivetoamines. proachhasbeenusedtotestforspecificityofendogenouslyex-
OurmixturedataindicatethatasamplingofOSNsthatexpressa pressedclassIIORs(Bozzaetal.,2002).
majorityoftheTAARsarepreferentiallyresponsivetoaminesbut We generated a strain of mice in which the T4 coding se-
canalsoexhibitsmallresponsestonon-amineswhenstimulated quence is replaced with that of a different receptor, human
athighconcentrations. TAAR5 (hT53T4–IRES–tauVenus). In these mice, OSNs that
wouldnormallyexpresstheT4receptorareforcedtoexpresshT5
TAAR-expressingOSNsarebroadlytunedtoamines proteininstead,andtheneuronsarelabeledwiththefluorescent
Next,weexaminedtheresponseprofilesofOSNsexpressingspecific markertauYFP(Fig.4A,B).OSNsexpressingthemodifiedallele
TAARs.Todothis,weusedmiceinwhichOSNsexpressingdefined (hT5–OSNs)arelocatedthroughoutthedorsalolfactoryepithe-
TAARgenesarelabeledwithfluorescentmarkersandtargetedthese lium,inapatternthatissimilartothatobservedinT4–RFPmice
cellsforpatch-clamprecording.Toexaminetheresponseproperties (compareFigs.3B,4B).Inaddition,axonsofOSNsexpressing
ofOSNsexpressingTAAR3(T3–OSNs),weusedtheTaar3–IRES– hT5coalesceintoglomeruliinthedorsalaspectofthemainol-
tauVenusstrain(Pacificoetal.,2012)inwhichOSNsexpress- factorybulb(Fig.4B),inthesameregionasaxonsexpressingT4
ing the endogenous Taar3 gene are labeled with a YFP (Fig. andotherTAARs(Pacificoetal.,2012).ThisindicatesthathT5
2A–C). T3–OSNs were tested with a set of six amines: functionsproperlytomediateaxonguidancewhenexpressedin
N-methylpiperidine, cyclohexylamine, (cid:1)-phenylethylamine, mice.
trimethylamine,isopentylamine,andcadaverine.Ofthese,T3has SwappingT4forhT5causedadramaticalterationinodorant
beenreportedtorespondspecificallytoisopentylamineandcyclo- responseprofile.T4–OSNsrespondedrobustlytoallofthetested
hexylamine(LiberlesandBuck,2006).Unexpectedly,T3–OSNsre- amineswhentestedat1(cid:2)M(n(cid:2)4cells;Fig.4C).Incontrast,
spondedtoallsixoftheamineswhenstimulatedat1(cid:2)M(n(cid:2)9cells; hT5–OSNsexhibitedonlysmallresponsestosomeoftheamines
Fig. 2D,E). The effective stimuli included primary and tertiary (trimethylamine,cyclohexylamine,andN-methylpiperidine)at
amines and a diamine. At 1 (cid:2)M, the most effective stimuli were 1 (cid:2)M (n (cid:2) 4 cells). No responses were seen at concentrations
isopentylamine and cyclohexylamine, with the latter eliciting the below 1 (cid:2)M (data not shown). These results indicate that the
largerresponses.Theaverageresponseamplitudewas107.3(cid:5)25.7 robustresponsestoalloftheaminesthatweobserveinT4–OSNs
pAforisopentylamineand157.6(cid:5)31.1pAforcyclohexylamine aremediatedsolelybytheexpressedT4protein.
Zhang,Pacificoetal.•UltrasensitiveAmineDetection J.Neurosci.,February13,2013•33(7):3228–3239•3231
Noligandshavebeenreportedprevi-
ously for human olfactory TAARs. To
identifyligands,wescreened40individual
amines on hT5–OSNs. Of these, hT5–
OSNs responded best to N,N-
dimethylethylamine, a structural analog
oftrimethylamine(Fig.4D).Whentested
across concentration, the observed EC
50
for N,N-dimethylethylamine was 1.2 (cid:5)
0.6(cid:2)Mwithathresholdof10(cid:6)7M(n(cid:2)6
cells). Our anatomical and physiological
data together indicate that hTAARs can
mediate both chemoreceptive and axon
guidancefunctionswhenexpressedinthe
mouseolfactorysystem.
TAAR4ishighlysensitivetoamines
Thelarge-amplituderesponsesobservedin
T4–OSNssuggestedthatT4mightbehighly
sensitive to amines. To examine this, we
stimulatedT4–OSNswithaminesatmulti-
ple concentrations (Fig. 5A). In fact, T4–
OSNs were strikingly sensitive. The
observedEC for(cid:1)-phenylethylaminewas
50
1.0(cid:5)0.6(cid:3)10(cid:6)12M,withresponsethresh-
oldsnear1(cid:3)10(cid:6)14M(n(cid:2)8cells;Fig.5B).
Eventheaminesthatwerenotreportedpre-
viouslyasT4ligandsincelllineselicitedre-
sponsesatlowconcentrations(Fig.5B).The
meanEC valuesforcyclohexylamineand
50
N-methylpiperidinewere7.7(cid:5)4.4(cid:3)10(cid:6)10
M(n(cid:2)4cells)and5.1(cid:5)1.7(cid:3)10(cid:6)10M(n(cid:2)
4cells),withthresholdsnear10(cid:6)11M.These
responsesaresixordersofmagnitudelower
thanthoseobservedincelllinesandfiveto
sixordersofmagnitudelowerthanwhatis
observed for OSNs expressing canonical
ORs(Grosmaitreetal.,2006,2009;Zhanget
al.,2012).
Next,weaskedwhetherOSNsexpress-
ing TAARs other than T4 respond to
amines at comparably low concentra-
tions.Todothis,weexaminedthesensi-
tivity of T3–OSNs using its preferred
ligands isopentylamine and cyclohexyl-
amine. T3–OSNs were very sensitive to
theseligandsandrespondedoverabroad
concentrationrange(Fig.5C).Themean
EC forisopentylaminewas1.5(cid:5)0.8(cid:3)
50
10(cid:6)8Mwithathresholdof10(cid:6)12M(n(cid:2)4
Figure1. TAAR-expressingOSNsrespondselectivelytoamines.A,DiagramoftheT4deletioninwhichthecodingsequencefor cells).Similarly,themeanEC50forcyclo-
TAAR4isreplacedwiththatofVenusYFP(yellow),followedbyaloxPsite(blacktriangle).Confocalimageoftheolfactory hexylaminewas2.7(cid:5)1.0(cid:3)10(cid:6)7Mwitha
turbinatesina(cid:8)T4–YFPhomozygousmouseshowingadorsaldistributionoflabeledcells.Scalebar,200(cid:2)m.B, threshold of 10(cid:6)9 M (n (cid:2) 5 cells). The
Schematicofalternategenechoicein(cid:8)T4–YFPOSNs.AnOSNthatchoosestoexpressthe(cid:8)T4–YFPallele(yellowbox) mean EC for cyclohexylamine was sig-
50
doesnotexpressafunctionalOR(yellowOSNwithgraycilia)andgoesontomakeasecondchoicethatisrestrictedtoa nificantlylowerforT4thanforT3(ttest,
subsetofTAARgenes.DiagramoftheTAARclusterwiththepotentiallychosengenesshownincolor.Non-olfactoryTaar1 p(cid:9)0.05).Thus,T3–OSNsarehighlysen-
isshowninwhite.Non-chosenTAARsareshowninblack.Asapopulation,YFP-labeledOSNscoexpressvariousTAARs
(yellowOSNswithcoloredcilia).Fluorescentcellsaretargetedforpatchrecording.C,Odor-evokedcurrentsina(cid:8)T4–OSN
stimulatedwith200mspulsesofodorantmixturescontainingamines,carboxylicacids,aldehydes,orketones.High
4
concentrations(1(cid:2)Meachodorant)wereusedtorevealthefullbreadthoftuning.Cellshownrespondedwithrobust
inwardcurrents(downwarddeflections)totheaminesmix,tothepositivecontrolphosphodiesteraseinhibitorIBMX,but thepeakresponseamplitude.E,Dotplotsshowingthere-
nottoothermixturesortosaline.Formixturecomponents,seeMaterialsandMethods.Actionpotentialtransientscanbe sponseprofilesofIBMX-responsiveOSNsfrom(cid:8)S50(n(cid:2)10
seenridingontopofthecurrenttraces.D,Dotplotsshowingtheresponseprofilesacrossodorantmixturesfor46 cells)and(cid:8)M72(n(cid:2)9cells)micethatexpressclassIand
IBMX-responsive (cid:8)T4–OSNs. Each row represents a single OSN. The area of the dot is proportional to classIIORs,respectively.
3232•J.Neurosci.,February13,2013•33(7):3228–3239 Zhang,Pacificoetal.•UltrasensitiveAmineDetection
sitivetoaminesbutlesssensitivethanT4-
OSNstoknownagonists.
TofurthertestthesensitivityofOSNsex-
pressingTAARsotherthanT4,werecorded
fromlabeledOSNsinhomozygous(cid:8)T4–YFP
mice.Intheseanimals,(cid:8)T4–OSNscoexpress
a random sample of the dorsally expressed
TAARgenes(Pacificoetal.,2012)exceptfor
T4,whichisabsentfromthegenome(Fig.1A).
Toassesssensitivityamongneuronsthathave
differentresponseprofiles,weusedamixture
of amines (trimethylamine, cadaverine,
N-methylpiperidine, (cid:1)-phenylethylamine,
isopentylamine, and cyclohexylamine) at
multipleconcentrations.Fiveof14homozy-
gous (cid:8)T4–OSNs responded to the amine
mixtureataconcentrationbelow1(cid:2)M(Fig.
5D).Twoexhibitedthresholdsinthepicomo-
larrangeandEC50values(cid:10)2nM.However,we
didnotobservecellsthatwereassensitiveas
T4–OSNsinthissample.Thus,oftheTAARs
thatwesampled,T4appearstobethemost
sensitive.
TAAR4-expressingOSNsusethe
canonicalodoranttransductioncascade
GiventhatTAAR-expressingOSNscom-
priseageneticallydistinctcellpopulation
intheolfactoryepithelium(Pacificoetal.,
2012)andthatT4–OSNsexhibitunusu-
ally high sensitivity, we tested whether
TAAR–OSNsmightsignalthroughadis-
tinct transduction pathway from typical
ORs.ORscoupletoanolfactory-specific
G-protein(G(cid:3) )thatgeneratesincreases
olf
incAMPviaanolfactory-enrichedadeny-
lyl cyclase (type III). Increases in cAMP
open cyclic nucleotide-gated channels
(CNGA2/CNGB1b), which in turn pass
Ca2(cid:7) ions that open calcium-gated Cl(cid:6)
channels, most likely anoctamin 2
(Kleene, 2008; Stephan et al., 2009). To
test for expression of olfactory signal
transductioncomponents,weperformed
4
indicatesthelocationofaloxPsite.B,Confocalimageofthe
olfactoryturbinatesinaT3–YFPheterozygousmouseshowing
labeledOSNsinthedorsalepithelium.Scalebar,200(cid:2)m.C,
Diagramoftheelectrophysiologicalrecordingconfigura-
tion. Odor-evoked currents are recorded via perforated
patchfromthedendriticknobsofOSNsinasemi-intact
preparationoftheolfactoryepitheliumwhileodorantsare
puffedontothecilia.D,Odor-evokedcurrentsmeasuredin
voltage-clampmodeinageneticallyidentifiedT3–YFP
OSN.Odorantswerepresentedfor200msat1(cid:2)M(n(cid:2)9
cells).T3–OSNsrespondedwithrobustinwardcurrents
(downwarddeflections)toodorantsbutnottosaline.Ac-
tionpotentialtransientscanbeseenridingontopofthe
currentrecordings.E,ResponseprofilesofT3–YFPOSNs
at10nM(n(cid:2)7cells)and1(cid:2)M(n(cid:2)9cells).Odorsare
Figure2. TAAR3-expressingOSNsarebroadlytunedtoamines.A,DiagramoftheTAARgeneclusterandtheTAAR3targeted trimethylamine(TMA),cadaverine(CAD),N-methylpiperidine
allele.Polygonsindicategeneorientation;olfactoryTAARsareshowninblack,andTaar1isshowninwhite.ThemodifiedTaar3 (NMP),(cid:1)-phenylethylamine(PEA),isopentylamine(IPA),and
locuscontainsaninternalribosomeentrysite(grayboxmarked“i”)andafluorescentmarkertauVenus(yellowbox).Blacktriangle cyclohexylamine(CHX).
Zhang,Pacificoetal.•UltrasensitiveAmineDetection J.Neurosci.,February13,2013•33(7):3228–3239•3233
combinedfluorescenceinsituhybridiza-
tion and immunohistochemistry on T4-
expressingOSNs.WefindthatT4–OSNs
express G(cid:3) , consistent with previous
olf
findings(LiberlesandBuck,2006).Inad-
dition,T4–OSNsexpressothermembers
of the canonical odorant transduction
cascade, including ACIII, CNGA2,
CNGB1b, PDE1c, PDE4a, Ric8b, and
Ano2(Fig.6A).
IfTAAR-expressingOSNsusetheca-
nonical odorant transduction cascade,
known modulators of this pathway
shouldinduceorblocktransductioncur-
rentinT4–OSNs.Infact,weobservethat
amineresponsesinT4–OSNsareblocked
by the adenylyl cyclase blocker MDL-
12,330A(Fig.6B).Thisblockissimilarto
what is observed for OSNs expressing a
canonical class II OR, M72 (Fig. 6B).
Thesedataarealsoconsistentwiththeob-
servationthatTAAR-expressingOSNsre-
spondtothephosphodiesteraseinhibitor
IBMX, which increases intracellular cAMP
(Fig.1Canddatanotshown).Thus,thehighly
sensitive amine responses in TAAR-
expressingOSNsaremostlikelymediated
by coupling to the canonical odorant
transductioncascade.
TAAR4glomeruliarebroadlytuned
andhighlysensitivetoamines
Next,weexaminedwhethertheresponse
propertiesobservedforT3andT4inour
invitroelectrophysiologicalassayarealso
observed in vivo when odorants are pre-
sented under more physiological condi-
tions in the vapor phase. To do this, we
imagedodor-evokedactivityfromgenet-
ically identified T3 and T4 glomeruli in
anesthetizedmice(Fig.7).Thiswasdone
using heterozygous OMP–spH mice in
which the activity reporter spH is ex-
pressedinallmatureOSNs(Bozzaetal.,
2004). Mice were also heterozygous for
the T4–RFP allele, the T3–YFP allele, or
both.Usingappropriatefluorescencefil-
ters,wecouldseparatethegreen,yellow,
andredfluorescencesignalsofspH,YFP,
and RFP, respectively (see Materials and
Methods),allowingustoidentifyT3and
T4glomeruliinthesamemice(Fig.7C).
4
Odorantswerepresentedfor200msat10nM.T4–OSNsre-
spondedwithrobustinwardcurrents(downwarddeflections)
toodorantsbutnottosaline.Spontaneousandodor-evoked
firingcanbeseenastransientsridingontopofthecurrent
traces.E,ResponseprofilesofT4–RFPOSNsat0.5nM(n(cid:2)9
Figure3. TAAR4-expressingOSNsarebroadlytunedandhighlysensitive.A,DiagramoftheTAARgeneclusterandtheTAAR4 cells)and10nM(n(cid:2)13cells).Odorsaretrimethylamine
targetedallele.AnnotationsareasdescribedinFigure2.B,ConfocalimageoftheolfactoryturbinatesinaT4–RFPhomozygous (TMA), cadaverine (CAD), N-methylpiperidine (NMP),
mouseshowinglabeledOSNsinthedorsalepithelium.Scalebar,200(cid:2)m.C,Odor-evokedcurrentsarerecordedfromfluorescently (cid:1)-phenylethylamine(PEA),isopentylamine(IPA),andcy-
labeledOSNsviaperforatedpatchfromthedendriticknobs.D,Odor-evokedcurrentsinageneticallyidentifiedT4–RFPOSN. clohexylamine(CHX).
3234•J.Neurosci.,February13,2013•33(7):3228–3239 Zhang,Pacificoetal.•UltrasensitiveAmineDetection
Asweshowedpreviously,theaminespref-
erentiallyactivatedglomeruliinthedorsal
caudal olfactory bulb (Pacifico et al.,
2012), including our genetically tagged
andneighboringglomeruli.
Consistent with our patch recordings,
T4glomeruliwerepreferentiallyresponsive
to (cid:1)-phenylethylamine at low concentra-
tionsbutwerealsobroadlytunedathigher
concentrations. In particular, isopentyl-
amine, cyclohexylamine, and cadaverine
elicitedrobustresponsesatnanomolarcon-
centrations (Fig. 7A). (cid:1)-Phenylethyla-
minewasbyfarthemostpotentactivatorof
T4glomeruli.Whentheamineswerepre-
sentedatafixedconcentration(2nM),T4
glomeruliexhibitedmuchlargerresponses
to (cid:1)-phenylethylamine than to any of the
otheramines(ANOVA,p(cid:9)0.001;Fig.7D).
T4glomeruliwerealsohighlysensitive
to amines. The lowest concentration at
which we obtained reliable responses to
(cid:1)-phenylethylamine was 100 pM (Fig.
7B,E),lowerthanthemostsensitiveglom-
eruli typically observed for canonical ORs
(Okaetal.,2006).Infact,T4glomeruliwere
theonlyglomeruliintheimagedregionof
thedorsalbulbthatrespondedtonanomo-
lar concentrations of (cid:1)-phenylethylamine
(Fig.7B;Pacificoetal.,2012).Perhapscon-
sistent with this high sensitivity, we also
notedthatT4glomeruliwerehighlysuscep-
tibletoadaptation.Evenwhenstimulatedat
low concentrations, response amplitudes
oftendecreasedsignificantlywithmultiple
odorant applications, making dose–re-
sponse experiments particularly challeng-
ing.Despitethis,itisclearthatT4glomeruli
are highly sensitive and preferentially re-
sponsiveto(cid:1)-phenylethylamine.
Our patch-clamp recordings showed
thatT4–OSNsweremoresensitivetothe
published T3 ligand cyclohexylamine
than T3–OSNs. To directly compare the
relativesensitivitiesofT3andT4invivo,
we recorded from mice in which both
glomeruliweredifferentiallylabeled.Con-
sistent with our in vitro data, cyclohexyl-
amineactivatedbothT3andT4glomeruli.
Interestingly, the thresholds observed for
both T3 and T4 in vivo were comparable
(Fig.7C,F),(cid:10)500pM.Themeanresponse
amplitudes across all concentrations were
Figure4. AmineresponsesaremediatedbytheexpressedTAAR4protein.A,DiagramoftheTAARgeneclusterandtargetedallele.
notsignificantlydifferentbetweenT3and
Polygonsindicategeneorientation;olfactoryTAARsareshowninblack,andTaar1isshowninwhite.TheTaar4locusisshownwithcoding
T4(ANOVA,p(cid:2)0.83).Thus,T4responds
sequence(whitebox),nontranslatedregions(blackboxes),andtranscriptionstart(arrow).ThehT53T4targetedinsertionswapsthe
toboth(cid:1)-phenylethylamineandcyclohexy- mouseT4codingsequenceforthatofhT5(violetbox),followedbyaninternalribosomeentrysite(grayboxmarked“i”)andthecoding
lamine in vivo despite the fact that cyclo- sequencefortauVenus(yellowbox).LoxPsiteisshown(blacktriangle).B,Left,MedialviewofolfactoryturbinatesinanhT53T4
hexylaminewasexcludedasaT4ligandby homozygousmouseshowingyellow-labeledOSNsandaxons.Right,DorsalviewoftheolfactorybulbsinanhT53T4heterozygousmouse
previousstudies(LiberlesandBuck,2006; showinglocationofaxonalconvergencetodorsalglomeruli.Scalebar,500(cid:2)m.C,AveragepeakcurrentelicitedbyodorantsinT4–RFP–
Ferreroetal.,2012).Infact,T4isatleastas OSNs(n(cid:2)4cells)andhT53T4–OSNs(n(cid:2)4cells)testedat1(cid:2)M.Stimuliaretrimethylamine(TMA),cadaverine(CAD),
sensitivetocyclohexylamineasT3,although N-methylpiperidine(NMP),(cid:1)-phenylethylamine(PEA),isopentylamine(IPA),cyclohexylamine(CHX),acetophenone(ACP),andN,N-
cyclohexylamineisoneofthetwomostpo- dimethylethylamine(DMEA).D,CurrentrecordingsfromasinglehT53T4–OSN(left)anddose–responsecurve(right)forN,N-
tentidentifiedligandsforT3.Together,our dimethylethylamine.Smoothlineisleast-squaresfitoftheHillequation,EC50(cid:2)1.2(cid:5)0.6(cid:2)M(n(cid:2)6cells).
Zhang,Pacificoetal.•UltrasensitiveAmineDetection J.Neurosci.,February13,2013•33(7):3228–3239•3235
TuningbreadthoftheTAARs
The response profiles observed in T3–
OSNsandT4–OSNsarenotablydifferent
from those observed with heterologous
expressionofthesamereceptors(Liberles
andBuck,2006).Weobservethattheli-
gandsidentifiedforT3andT4inheterol-
ogouscellswerethemosteffectivestimuli
for T3–OSNs and T4–OSNs. However,
theseOSNsalsorespondedtostructurally
diverseamines,particularlyathighercon-
centrations. Responses to these unex-
pectedligandsarelikelyrelevantforodor
codingbecausetheycouldbeobservedas
increasesinactionpotentialfiringratesin
singleOSNsandbyglomerularactivation
inintactmice.
Onepotentialexplanationforthediffer-
encesintuningbreadthobservedusingour
invivoexpressionapproachisthatthege-
neticallytaggedOSNscouldexpressother
receptors.However,therobustresponsesto
all of the effective ligands were abolished
whenT4wasreplacedwithanotherrecep-
tor,indicatingthatthesingle,identifiedre-
ceptormediatesalloftheobservedodorant
responses. Analyzing a receptor swap is
preferabletoanalyzingareceptordeletion
becauseOSNsthatexpressaTAARdele-
tion allele go on to express other TAAR
genes(Johnsonetal.,2012;Pacificoetal.,
2012).Nevertheless,wedidanalyzeapopula-
tionofhomozygous(cid:8)T4–OSNsandfound
thattheyrarelyrespondtocyclohexylamine
and N-methylpiperidine, consistent with
theideathatT4mediatesresponsestothese
compoundsaswell(datanotshown).Our
observationsarguestronglythattheamine
responsesobservedinourgeneticallytagged
Figure5. TAAR-expressingOSNsarehighlysensitivetoamines.A,CurrentrecordingsfromageneticallyidentifiedT4–RFPOSN
stimulatedwith(cid:1)-phenylethylamine(PEA)atmultipleconcentrations(200mspulses).T4–OSNsrespondedwithrobustinward TAAR–OSNsaremediateddirectlybyasin-
currents(downwarddeflections).B,Dose–responserelationshipofT4–RFPOSNstoN-methylpiperidine(NMP;n(cid:2)4cells), gleexpressedreceptor.
(cid:1)-phenylethylamine(PEA;n(cid:2)8cells),andcyclohexylamine(CHX;n(cid:2)4cells).C,Averagedose–responsedatafromT3–OSNsfor Themostlikelyexplanationforthedif-
isopentylamine(IPA;n(cid:2)4cells)andcyclohexylamine(CHX;n(cid:2)5cells).Additionaldataathigherconcentrationswereusedto ferencebetweenourdataandpreviousre-
fittheHillfunction,butthegraphiscroppedtomatchtherangeofBandDtoallowdirectcomparison.D,Individualdose–response sults is that the TAARs exhibit different
relationshipfrom14(cid:8)T4–OSNsfromhomozygous(cid:8)T4–YFPmice.OSNswerestimulatedwithmultipleconcentrationsofan response profiles—or are simply more
aminesmixcontainingtrimethylamine,cadaverine,N-methylpiperidine,(cid:1)-phenylethylamine,propylamine,andcyclohexyl- sensitive—whenexpressedinOSNscom-
amine.Becauseofadaptationinsomecells,dataarefromthelargestresponsestoeachstimulus.Smoothlinesareleast-squaresfits
paredwithculturedcells(Shirokovaetal.,
oftheHillequation.
2005;VonDanneckeretal.,2005;Okaet
al., 2006; Grosmaitre et al., 2009). OSNs
dataindicatethatT4hasarelativelyhighsensitivitytoaminesinvivo likely have specialized mechanisms for
aswellasinvitro,evencomparedwithotherTAARs. proper expression and localization of receptors (Matsunami et
al.,2009).ThereadoutofTAARactivationmaybemoresensitive
Discussion inOSNsbecausethereceptorsarelocalizedathighdensityincilia
Herewereport,forthefirsttime,theodorantresponsespecificityof andarecoupledtotheendogenoussignaltransductioncascade.
OSNs expressing genetically identified TAARs and their corre- Consequently,recordingfromthenativecelltypeallowsustode-
spondingglomeruliinmice.WefindthatTAAR-expressingOSNs tecteffectiveligandsthataremissedinotherassays.Ourdatasupport
areselectivelyactivatedbyamines,arebroadlytunedathighercon- thisview,becausetheresponsethresholdfor(cid:1)-phenylethylaminein
centrations,andexhibitresponsethresholdsthatareseveralor- T4–OSNswassub-picomolar:1,000,000-foldlowerthanthepreviously
ders of magnitude lower than any previously published main reportedT4thresholdsfromculturedcells(Ferreroetal.,2011).
olfactory chemoreceptor. This unprecedented sensitivity is most
likelymediatedbycouplingtothecanonicalodoranttransduction SensitivityoftheTAARs
pathway.Ourdataprovideinsightintotheuniquecontributionof Inourexperiments,wehavemeasuredthesensitivityofT3,T4,
theTAARstovertebrateolfaction. hT5, and a subset of randomly chosen mouse TAARs (from
3236•J.Neurosci.,February13,2013•33(7):3228–3239 Zhang,Pacificoetal.•UltrasensitiveAmineDetection
Figure6. TAAR4–OSNsusethecanonicalodoranttransductioncascade.A,Combinedimmunohistochemistryandfluorescenceinsituhybridizationforknownolfactorysignaltransduction
componentgenesinsectionsoftheolfactoryepithelium.T4–OSNswerevisualizedinT4–ChR–YFPmice(Pacificoetal.,2012)bystainingwithanantibodytoGFP(green).Insituhybridizationis
shownforeightprobes(red).NucleiarestainedwithTOPRO-3(blue).Foreachpairofpanels,theleftshowsthecombinedchannels,andtherightshowstheinsitusignalonly.Arrowsmarklocations
ofT4–OSNs.B,ReversibleblockofodorantresponsesinT4–OSNsusingtheadenylylcyclaseblockerMDL-12,330Aat30(cid:2)M(MDL).Responseto200mspulseof1nM(cid:1)-phenylethylamine(PEA;n(cid:2)
4OSNs)wascompletelyblockedandpartlyrecoveredafterwashout(left).AsimilarblockwasseenforOSNsexpressingacanonicalclassIIORM72(n(cid:2)3)stimulatedwith200mspulsesof100(cid:2)M
acetophenone(ACP;right).Dataforsinglecellsareshowningrayandaveragedatainblack.Insetsaboveshowindividualcurrenttraces.
Zhang,Pacificoetal.•UltrasensitiveAmineDetection J.Neurosci.,February13,2013•33(7):3228–3239•3237
Figure7. TAAR4glomeruliarehighlysensitiveandbroadlytunedinvivo.A,Opticalimagingofodor-evokedspHsignalsfromglomeruliinthedorsal,caudalolfactorybulbofaT4–RFP
heterozygousmouse.RestingspHfluorescence(greenpanel)showsglomeruli,andRFPfluorescence(redpanel)showsonelargeT4glomerulus(largearrowhead)andonesmallerT4glomerulus
(smallerarrowhead).Pseudocoloredpanelsshowresponsesto(cid:1)-phenylethylamine(PEA),trimethylamine(TMA),andcadaverine(CAD)attheindicatedvaporconcentrations.B,Opticalimaging
ofresponsesinT4glomeruliofaheterozygousT4–RFPmouseelicitedwithdifferentconcentrationsofPEA.RestingspHfluorescence(greenpanel)showsglomeruliinthedorsalolfactorybulb.RFP
fluorescence(redpanel)showsalabeledT4glomerulus(arrowhead).Pseudocoloredpanelsshowodor-evokedchangesinfluorescence((cid:8)F)inresponsetoincreasingconcentrationsof
(cid:1)-phenylethylamine(PEA).C,SimultaneousrecordingfromT3andT4glomeruliinthesamemouse.TopshowsrestingfluorescencefromT4–RFP(whitearrowhead)andT3–YFP(grayarrowhead)
glomeruli.Bottomthreepanelsshowresponsestovaryingconcentrationsofcyclohexylamine(CHX).Inthisanimal,T3exhibitedlargerresponsesatthreshold(500pM).ThehighestCHX
concentrationelicitedalargeresponseinT3andT4,aswellasamoremedial,neighboringglomerulusthatmostlikelycorrespondstoanotherTAARgene.D,AverageresponseamplitudeofT4
glomerulitoasetofaminesat2nMvaporconcentration(n(cid:2)6glomerulifrom6mice):(cid:1)-phenylethylamine(PEA),cyclohexylamine(CHX),trimethylamine(TMA),cadaverine(CAD),and
N-methylpiperidine(NMP).E,AverageresponseamplitudeofT4glomerulitomultipleconcentrationsofPEA(n(cid:2)10glomerulifrom10mice).F,AverageresponseamplitudesofT3glomeruli
(green)andT4glomeruli(red).ToimprovesensitivitytoCHX,theserecordingsweremadewiththecontralateralnarisoccluded,whichincreasesflowrateandproduceslargerresponses.Dataare
fromfiveT3/T4compoundheterozygousmice,twoT4–RFPmice,andoneT3–YFPmouse.Dataaremean(cid:5)SEM.InA–C,anterior(a)isup,medial(m)istotheleft,andresponsesarescaledtothe
samemaximum(cid:8)F.Scalebar,200(cid:2)m.