Sensory Neurons Arouse C. elegans Locomotion via Both Glutamate and Neuropeptide Release

20 Pages·2015·3.06 MB·English

RESEARCHARTICLE
Sensory Neurons Arouse .
C elegans
Locomotion via Both Glutamate and
Neuropeptide Release
SeungwonChoi1,2,3☯,KelseyP.Taylor1,2,3☯,MariosChatzigeorgiou4,ZhitaoHu1,2,
WilliamR.Schafer4,JoshuaM.Kaplan1,2,3*
1 DepartmentofMolecularBiology,MassachusettsGeneralHospital,Boston,Massachusetts,UnitedStates
ofAmerica,2 DepartmentofNeurobiology,HarvardMedicalSchool,Boston,Massachusetts,UnitedStates
ofAmerica,3 BiologicalandBiomedicalSciencesprogram,HarvardMedicalSchool,Boston,
a11111 Massachusetts,UnitedStatesofAmerica,4 CellBiologyDivision,MRCLaboratoryofMolecularBiology,
Cambridge,UnitedKingdom
☯Theseauthorscontributedequallytothiswork.
* [email protected]
Abstract
OPENACCESS
Citation:ChoiS,TaylorKP,ChatzigeorgiouM,HuZ, C.elegansundergoesperiodsofbehavioralquiescenceduringlarvalmolts(termedlethar-
SchaferWR,KaplanJM(2015)SensoryNeurons gus)andasadults.Littleisknownaboutthecircuitmechanismsthatestablishthesequies-
ArouseC.elegansLocomotionviaBothGlutamate
centstates.Lethargusandadultlocomotionquiescenceisdramaticallyreducedinmutants
andNeuropeptideRelease.PLoSGenet11(7):
e1005359.doi:10.1371/journal.pgen.1005359 lackingtheneuropeptidereceptorNPR-1.Here,weshowthatthearousedlocomotionof
npr-1mutantsresultsfromtheexaggeratedactivityinmultipleclassesofsensoryneurons,
Editor:AndrewD.Chisholm,UniversityofCalifornia
SanDiego,UNITEDSTATES includingnociceptive(ASH),touchsensitive(ALMandPLM),andstretchsensing(DVA)
neurons.Thesesensoryneuronsacceleratelocomotionviabothneuropeptideandgluta-
Received:January30,2015
materelease.Therelativecontributionofthesesensoryneuronstoarousaldiffersbetween
Accepted:June13,2015
larvalmoltsandadults.Ourresultssuggestthatabroadnetworkofsensoryneuronsdic-
Published:July8,2015 tatestransitionsbetweenarousedandquiescentbehavioralstates.
Copyright:©2015Choietal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
AuthorSummary
medium,providedtheoriginalauthorandsourceare
credited.
Animalsswitchbetweenperiodsofbehavioralarousalandquiescenceinresponsetoenvi-
DataAvailabilityStatement:Allrelevantdataare ronmental,developmental,andcircadiancues.Littleisknownaboutthecircuitmecha-
withinthepaperanditsSupportingInformationfiles. nismsthatproducethesebehavioralstates.Duringlarvalmolts,C.elegansexhibitsasleep-
Funding:ThisworkwassupportedbyPredoctoral likestate(termedlethargus)thatischaracterizedbytheabsenceoffeedingandprofound
FellowshipsfromtheKwanjeongEducational locomotionquiescence.Wepreviouslyshowedthatmutantslackingtheneuropeptide
Foundation(SC)andtheNSF(KPT)(https://www. receptorNPR-1exhibitincreasedarousalduringlarvalmolts,whichisinpartmediatedby
nsfgrfp.org),andbyresearchgrantstoJMK(NIH
increasedsecretionofanarousalpeptide(PDF-1).Here,wecomparethecircuitsregulat-
DK80215)(grants.nih.gov)andWRS(MRCMC-
ingarousalinlarvalmoltsandadults.Weshowthatabroadnetworkofsensoryneurons
A022-5PB9).(www.mrc.ac.uk).Thefundershadno
arouseslocomotionbutthattheimpactofeachneurondiffersbetweenlethargusand
roleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript. adults.WeproposethatthisbroadsensorynetworkallowsC.eleganstoadaptitsbehavior
acrossabroadrangeofdevelopmentalandphysiologicalcircumstances.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
Introduction
Animalsundergoperiodsofbehavioralquiescenceandarousalinresponsetochangesintheir
environmentandmetabolicstate.Arousalisdefinedasastateofheightenedresponsivenessto
externalstimulicoupledwithincreasedmotoractivitywhereasquiescenceisassociatedwith
diminishedresponsivenessandmotoractivity[1].Quiescenceandarousalcanpersistformin-
utestohours.Arousalisassociatedwithfear,stress,hunger,andexposuretosexualpartners
[1],whilequiescenceisassociatedwithsleepandsatiety[2].Relativelylittleisknownaboutthe
specificcircuitmechanismsleadingtoarousalorquiescence.Inparticular,itisunclearifsimi-
larmechanismsmediatequiescenceandarousalinresponsetodifferentcues,oratdifferent
timesduringdevelopment.Toaddressthisquestion,wehaveanalyzedarousalandquiescence
ofC.eleganslocomotion.
Duringeachlarvalmolt,C.elegansundergoesaprolongedperiodofprofoundbehavioral
quiescence,termedlethargusbehavior,wherebylocomotionandfeedingbehaviorsareinac-
tiveforapproximately2hours[3].Lethargushaspropertiesofasleep-likestatesuchas
reducedsensoryresponsivenessandhomeostaticreboundofquiescencefollowingperturba-
tion[4].Severalgenesandmolecularpathwaysinvolvedinlethargusbehaviorhavebeen
identified[4–11].Multiplesensoryresponsesarediminishedduringlethargus,including
thosemediatedbyanociceptiveneuron(ASH)[12],andbymechanosensoryneurons
[11,13].
MutantslackingNPR-1NeuropeptideY(NPY)receptorshavebeenutilizedasamodelfor
generalizedarousal.NPR-1inhibitstheactivityofacentralsensorycircuitthatisdefinedby
gapjunctionstotheRMGinterneuron[14].Innpr-1mutants,responsesmediatedbythe
RMGcircuit(e.g.pheromoneandoxygenavoidance)areexaggerated,andthisheightened
acuityisassociatedwithexaggeratedlocomotion(bothduringlethargusandinadults)
[11,14–16].Mutationsthatincrease(e.g.npr-1)anddecrease(e.g.tax-4CNGandosm-9
TRPV)RMGcircuitactivityareassociatedwithlocomotionarousalandquiescencerespec-
tively[11,14,17,18].
Wepreviouslyshowedthatlocomotionquiescenceduringlethargusisdramatically
reducedinnpr-1mutantsandthatthiseffectrequiresincreasedRMGsensoryactivity[11].
Subsequentstudiesshowedthatinmicrofluidicchambersnpr-1mutantshavemodestdefects
inlethargusquiescencewhensensorycuesareminimizedbutthatdramaticquiescencedefects
areobservedfollowingbriefstimulationwithlightorvibration[19,20].Takentogether,these
paperssuggestthatnpr-1mutantsexhibitarousedlocomotionasaconsequenceofenhanced
sensoryactivity.
ThearousingeffectsoftheRMGcircuitaremediatedinpartbysecretionofaneuropeptide,
pigmentdispersingfactor(PDF-1)[11].ActivationofPDFreceptors(PDFR-1)inperipheral
mechanosensoryneuronsenhancessensitivitytovibration,therebyacceleratinglocomotion.
Thus,sensoryevokedactivityintheRMGcircuitarouseslocomotionduringlethargusthrough
changesinPDF-1andPDFR-1signaling.Theseresultsraiseseveralinterestingquestions.
Whichspecificsensoryneuronsareresponsibleforarousal?DoestheRMGcircuitregulate
arousalviamultipleoutputs(i.e.inadditiontoPDF-1)?DoestheRMGcircuitfunctionsimi-
larlyduringlethargusandinadults?Isdiminishedsensoryacuityduringlethargusrequiredfor
behavioralquiescence?
Hereweshowthatglutamatergictransmissionpromotesarousal,weidentifyglutamatergic
neuronsandglutamatereceptorsthatmediatearousal,andweshowthatarousaloccursbydis-
tinctmechanismsinlethargusandadultanimals.
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
Results
CholinergictransmissionatNMJsisincreasedinnpr-1adults
Adultnpr-1mutantsexhibitacceleratedlocomotion(Fig1A–1C),asshowninpriorstudies
[21].Fasteradultlocomotionsuggeststhatlocomotioncircuitactivityhasbeenaltered.Consis-
tentwiththisidea,npr-1mutantadultshaveenhancedsensitivitytotheparalyticeffectsofa
cholinesteraseinhibitor(aldicarb)(Fig1D–1FandS2AFig)[22],indicatingincreasedexcit-
atorytransmissionatneuromuscularjunctions(NMJs).Tomoredirectlyassesschangesin
synaptictransmission,werecordedminiatureexcitatorypost-synapticcurrents(mEPSCs)in
bodymuscles,whichareevokedbyacetylcholine(ACh)releaseatNMJs.ThemEPSCrate
observedinnpr-1adultswassignificantlyhigherthaninwildtypecontrolswhilemEPSC
amplitudeswereunaltered(Fig1G–1I).FastermEPSCratessuggestthatAChreleasefrom
motorneuronswasincreasedwhereasunalteredmEPSCamplitudesimplythatmusclerespon-
sivenesstosecretedAChwasunaffected.Bycontrast,neitherAChreleaseevokedbydepolariz-
ingmotorneuronswithastimulatingelectrode(evokedEPSCs),nortransmissionat
GABAergicNMJs(assessedbyminiatureinhibitorypost-synapticcurrents,mIPSCs)was
alteredinnpr-1mutants(S1Fig).Thisconstellationofelectrophysiologicaldefectssuggests
thattonicAChrelease(assessedbymEPSCrate)wasenhancedinnpr-1mutants,whereas
otherformsofneurotransmitterrelease(evokedAChreleaseandtonicGABArelease)were
unaffected.EnhancedtonicAChreleaseatNMJscouldaccountfortheacceleratedlocomotion
rateobservedinnpr-1adults.
Enhancedcholinergictransmissioninnpr-1adultsiscausedby
increasedsensoryactivity
Priorstudiesshowedthatseveralbehavioralphenotypesexhibitedbynpr-1mutantsarecaused
byenhancedsensitivitytoenvironmentalcues.Inparticular,sensoryresponsesmediatedby
theRMGcircuitareenhancedinnpr-1mutants[14,17,18]andthisenhancedsensoryacuityis
requiredforacceleratedlocomotionratesduringlethargus[11,20].Wedidseveralexperiments
todetermineifenhancedRMGcircuitactivityisalsorequiredforincreasedcholinergictrans-
missioninnpr-1adults.Atransgenerestoringnpr-1expressionintheRMGcircuit(usingthe
flp-21promoter)rescuedtheacceleratedlocomotion(Fig1B),enhancedaldicarbsensitivity
(Fig1DandS2AFig),andfastermEPSCrate(Fig1G–1I)defectsofnpr-1adults.Bycontrast,
annpr-1transgeneexpressedinGABAergicneuronslackedrescuingactivity(Fig1D–1H).
TheseresultsindicatethatNPR-1actsintheRMGcircuittoslowadultlocomotion.Similarly,
mutationsinactivatingionchannelsrequiredforsensorytransduction(TAX-4/CNGand
OCR-2/TRPV)intheRMGcircuitsuppressedthenpr-1adultlocomotion(Fig1C),aldicarb
sensitivity(Fig1Eand1FandS2BandS2CFig),andmEPSCrate(Fig1Jand1K)defects.Col-
lectively,theseresultssuggestthattheacceleratedadultlocomotionexhibitedbynpr-1mutants
iscausedbyheightenedactivityintheRMGsensorycircuitand,consequently,correspondsto
anarousedstate.
InactivatingPDFsignalingdoesnotpreventarousedlocomotioninnpr-1
adults
Wepreviouslyshowedthatthelethargusquiescencedefectsexhibitedbynpr-1mutantsare
causedbyincreasedsecretionofPigmentdispersingfactor(PDF-1)bycellsintheRMGcircuit
[11].BecausePDF-1secretionisalsoincreasedinnpr-1adults[11],wetestedtheideathatthe
hyperactiveadultlocomotionofnpr-1mutantsisalsocausedbyincreasedPDFsignaling.Con-
trarytothisidea,wefoundthatpdf-1andpdfr-1(PDFReceptor-1)mutationsreducedbutdid
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
Fig1.CholinergictransmissionatNMJsisenhancedbyincreasedsensoryactivityinnpr-1adults.Locomotionbehaviorofsingleadultwormswas
analyzedfortheindicatedgenotypes.Instantaneouslocomotionvelocity(A)andaveragelocomotionvelocity(B-C)areplotted.(A-C)Thenpr-1adult
locomotiondefectwasrescuedbytransgenesexpressingNPR-1intheRMGcircuit(RMGrescue,flp-21promoter),andsuppressedindoublemutants
lackingTAX-4/CNGchannels.(D-F)Thepercentageofanimalsparalyzedon1mMaldicarbat80minwereplottedfortheindicatedgenotypes.Thenumber
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
oftrialsisindicatedforeachgenotype.Fulltimecoursesofaldicarb-inducedparalysisareshowninS2A–S2CFig.(D)Thenpr-1aldicarbhypersensitivity
wasrescuedbytransgenesexpressingNPR-1intheRMGcircuit(RMGrescue,flp-21promoter)butnotbythoseexpressedinGABAergicneurons(GABA
rescue,unc-25andunc-30promoters).(E-F)Thenpr-1aldicarbhypersensitivitywasblockedbymutationsinactivatingTAX-4/CNGchannelsorOCR-2/
TRPVchannels.(G-L)mEPSCswererecordedfrombodywallmusclesofadultwormsfortheindicatedgenotypes.RepresentativetracesofmEPSCs(G
andJ)andsummarydataareshown(H,I,K,andL).(G-I)Thenpr-1cholinergictransmissiondefectwasrescuedbytransgenesexpressingNPR-1inthe
RMGcircuit(RMGrescue,flp-21promoter)butnotbythoseexpressedinGABAergicneurons(GABArescue,unc-30promoter).(J-L)Thenpr-1cholinergic
transmissiondefectwasabolishedbymutationsinactivatingTAX-4orOCR-2.Thenumberofanimalsanalyzedisindicatedforeachgenotype.Errorbars
indicateSEM.Valuesthatdiffersignificantlyareindicated(*,p<0.05;**,p<0.01;***,p<0.001;ns,notsignificant).
doi:10.1371/journal.pgen.1005359.g001
noteliminatethealdicarbhypersensitivity(Fig2Aand2BandS2DandS2EFig),theacceler-
atedlocomotion(Fig2C),andincreasedmEPSCrate(Fig2Dand2E)defectsofnpr-1adults.
Collectively,theseresultssuggestthatadditionalexcitatoryoutputsfromtheRMGcircuit(i.e.
beyondPDF-1)mustcontributetothearousedlocomotionofnpr-1adults.
Glutamatereleasedbysensoryneuronsisrequiredfornpr-1locomotion
andEPSCdefects
ManyC.eleganssensoryneuronsareglutamatergic,includingtwoneuronsintheRMGcircuit
(ASHandASK)andthebodytouchneurons[23].Todetermineifglutamatereleasebysensory
Fig2.InactivatingPDFsignalingdoesnotpreventarousedlocomotioninnpr-1adults.(A-B)Thenpr-1aldicarbhypersensitivitywasdecreasedbut
notabolishedbymutationsinactivatingPDF-1orPDFR-1.Thepercentageofanimalsparalyzedon1mMaldicarbat80minwereplottedfortheindicated
genotypes.Thenumberoftrialsisindicatedforeachgenotype.Fulltimecoursesofaldicarb-inducedparalysisareshowninS2DandS2EFig.(C)
Locomotionbehaviorofsingleadultwormswasanalyzedfortheindicatedgenotypes.Thenpr-1adultlocomotiondefectwasnotblockedbymutations
inactivatingPDF-1orPDFR-1.(D-F)mEPSCswererecordedfrombodywallmusclesofadultwormsfortheindicatedgenotypes.Representativetracesof
mEPSCs(D)andsummarydataareshown(E-F).Thenpr-1cholinergictransmissiondefectwasnotsuppressedbymutationsinactivatingPDFR-1.The
numberofanimalsanalyzedisindicatedforeachgenotype.ErrorbarsindicateSEM.Valuesthatdiffersignificantlyareindicated(*,p<0.05;**,p<0.01;***,
p<0.001;ns,notsignificant).
doi:10.1371/journal.pgen.1005359.g002
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
neuronsisrequiredforacceleratedlocomotioninnpr-1mutants,weanalyzedmutationsthat
inactivatethevesicularglutamatetransporter(eat-4VGLUT),whichisprimarilyexpressedin
sensoryneurons[23].eat-4VGLUTmutationsblockedtheincreasedmotilefractionandloco-
motionspeedofnpr-1mutantsbothduringtheL4-Adult(L4/A)molt(Fig3A–3C)andin
adults(Fig3Dand3E).eat-4mutationsalsoblockedthehypersensitivitytoaldicarb(Fig3F
Fig3.Glutamatereleasedbysensoryneuronsisrequiredforthenpr-1locomotionandthecholinergictransmissiondefects.Locomotionbehavior
ofsinglewormsduringtheL4/Alethargus(A-C)andinadults(D-E)wasanalyzedintheindicatedgenotypes.Instantaneouslocomotionvelocity(A,D),
averagemotilefraction(B),andaveragelocomotionvelocity(C,E)areplotted.Thenpr-1locomotiondefectwassuppressedbymutationsinactivatingEAT-
4/VGLUT,andpartiallyreinstatedbytransgenesexpressingEAT-4inASHneurons(sra-6promoter)andtouchneurons(mec-4promoter)ineat-4;npr-1
doublemutantsusingtheindicatedpromoters.AnEAT-4transgeneexpressedinASKneurons(sra-9promoter)lackedrescuingactivity.(F)Thenpr-1
aldicarbhypersensitivitywassuppressedbymutationsinactivatingEAT-4/VGLUT.Thepercentageofanimalsparalyzedon1mMaldicarbat80minwere
plottedfortheindicatedgenotypes.Thenumberoftrialsisindicatedforeachgenotype.Fulltimecoursesofaldicarb-inucedparalysisareshowninS2FFig.
(G-I)Thenpr-1cholinergictransmissiondefectwasabolishedbymutationsinactivatingEAT-4/VGLUT.mEPSCswererecordedfrombodywallmusclesof
adultwormsfortheindicatedgenotypes.RepresentativetracesofmEPSCs(G)andsummarydataareshown(H-I).Thenumberofanimalsanalyzedis
indicatedforeachgenotype.ErrorbarsindicateSEM.Valuesthatdiffersignificantlyareindicated(*,p<0.05;**,p<0.01;***,p<0.001;ns,notsignificant).
doi:10.1371/journal.pgen.1005359.g003
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
andS2FFig)andincreasedmEPSCrate(Fig3Gand3H)defectsofnpr-1adults.Transgenes
restoringEAT-4expressionintouchneuronsandASHneuronspartiallyreinstatedboth
lethargus(Fig3Band3C)andadultlocomotion(Fig3Dand3E)defectsineat-4;npr-1double
mutants,whereastransgenesexpressedinASKlackedrescuingactivity(Fig3Band3C).eat-4
transgeneshadnoeffectonlethargusquiescenceinwildtypeanimals(S3Fig).Theseresults
suggestthatglutamatereleasedbyASHandtouchneuronsarouseslocomotioninL4/Aand
adultnpr-1mutants.
ASHactivityisassociatedwithlocomotionarousal
TheprecedingresultssuggestthatASHsynapticoutputarouseslocomotioninnpr-1mutants.
Wedidseveraladditionalexperimentstotestthisidea.IfalteredASHoutputwererequiredfor
arousedlocomotion,wewouldexpectthatnpr-1mutantslackingASHneuronswouldhave
increasedlocomotionquiescence.Totestthisidea,weinducedASHcelldeathwithatransgene
thatexpressesthepro-apoptoticcaspaseCED-3.KillingASHsignificantlydecreasedtheL4/A
motilefractionandlocomotionrateinnpr-1mutants(Fig4A–4C).Bycontrast,ASHablation
hadlittleeffectonthelocomotionrateofnpr-1adults(Fig4D).
TodetermineifASHactivityisincreasedinnpr-1mutantsduringlethargus,weexamined
sensory-evokedcalciumresponsesinASH,usingthegeneticallyencodedcalciumindicator
Cameleon.ASHmediatesavoidanceresponsestocopperandhyper-osmoticstimuli.Consis-
tentwitharecentstudy[12],themagnitudeofcopper(Fig4Eand4F)andglycerol-evoked
(S4AandS4BFig)calciumtransientsinASHwassignificantlydecreasedduringlethargusin
wild-typeanimals.DecreasedASHresponsivenesstocopperandglycerolduringL4/Alethar-
guswasblockedinnpr-1mutants,whereasASHresponsivenessinadultswasunalteredinnpr-
1mutants(Fig4Eand4FandS4AandS4BFig).TransgenesexpressingNPR-1intheRMGcir-
cuit(usingtheflp-21promoter)orinASH(usingthesra-6promoter)reinstatedtheL4/A
decreaseincopperandglycerol-evokedASHcalciumtransientsinnpr-1mutants(Fig4Gand
4HandS4CandS4DFig).TheseresultssuggestthatNPR-1actsinASHtoinhibitsensory
responsesandthatincreasedASHactivityisrequiredforacceleratedlocomotionofnpr-1
mutantsduringlethargusbutnotinadults.
TodetermineifincreasedASHactivityissufficienttoarouselocomotion,weanalyzedloco-
motionafterartificiallydepolarizingASHneurons.Forthisexperiment,weutilizedtransgenic
animalsthatexpressratTRPV1capsaicinreceptorsinASHneurons[24].Intheseanimals,
capsaicintreatmentevokesASH-mediatedavoidancebehaviors[24].A5-hourcapsaicintreat-
menthadlittleeffectonL4/Amotilefractionandlocomotionvelocity[11],whereascapsaicin
treatmentsignificantlyacceleratedadultlocomotionandincreasedaldicarbsensitivity(Figs
4I–5JandS2GFig).TheseeffectswerenotobservedinanimalslackingTRPV1expressionin
ASHneurons(Fig4Iand4J).Thus,forcedASHdepolarizationwassufficienttoarouseadult
butnotletharguslocomotion.Collectively,theseresultssuggestthatdiminishedandheight-
enedASHactivityisassociatedwithlocomotionquiescenceandarousalrespectively;however,
themagnitudeofASH’sarousingeffectsdifferbetweenlethargusandadultanimals.
GLR-2AMPAreceptorsarerequiredforthenpr-1lethargusdefect
Whichglutamatereceptorsarouselocomotioninnpr-1mutants?Glutamate-activatedcation
channels,AMPA(GLR-1and-2)andNMDA(NMR-1and-2)receptors,mediateexcitatory
transmissionatASH-interneuron[25–27].Thenpr-1L4/Aquiescencedefectwasabolishedin
glr-2;npr-1doublemutants(Fig5A–5C),whileglr-1mutationshadnoeffect(Fig5Dand5E).
Bycontrast,glr-1,glr-2,andnmr-1mutationshadlittleeffectonnpr-1adultlocomotion(Fig
5FandS5Fig).Similarly,glr-2mutationsdidnotblocktheincreasedmEPSCrateinnpr-1
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
Fig4.ASHactivityisassociatedwithlocomotionarousal.LocomotionbehaviorduringtheL4/Alethargus(A-C)andinadults(D)ofsinglewormswhose
ASHneuronswereablatedbytransgenicoverexpressionofCED-3inASHneurons(sra-6promoter)wasanalyzedintheindicatedgenotypes.Animalswere
analyzedbyfluorescencemicroscopyafterlocomotionrecordingstodetermineifASHneuronswereablated(1–2ASH:animalswith1or2ASHintact
neurons;0ASH:animalslackingviableASHneurons).Instantaneouslocomotionvelocity(A),averagemotilefraction(B),andaveragelocomotionvelocity
(C-D)areplotted.Thenpr-1locomotiondefectduringtheL4/Alethargus,butnotinadults,waspartiallysuppressedinthetransgenicanimalsinwhichbothof
ASHneuronswereablated(0ASH).(E-H)Copper-evokedcalciumtransientsinASHwereanalyzedinL4,L4/A,andadultsoftheindicatedgenotypesusing
cameleonasacalciumindicator.Averagedresponses(E,G),andtheamplitudesofindividualtrials(F,H)areshownforeachgenotype.Eachtrace
representstheaveragepercentagechangeinYFP/CFPfluorescenceratio.Thelighttanrectangleindicatesthedurationforwhich10mMcopperwas
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GlutamateandNeuropeptidesArouseC.elegansLocomotion
applied.DarkgrayshadingofeachtraceindicatesSEMofthemeanresponse.(E-F)Copper-evokedcalciumtransientsinASHneuronsweresignificantly
reducedduringL4/Alethargus,andthiseffectwasabolishedinnpr-1mutants.(G-H)ThisdefectduringL4/Aletharguswasrescuedbytransgenes
expressingNPR-1intheRMGcircuit(RMGrescue,flp-21promoter)orinASHneurons(ASHrescue,sra-6promoter).(I-J)ForceddepolarizationofASH
neuronsincreasedadultlocomotionvelocity(I)andaldicarbsensitivity(J).RatTRPV1wasectopicallyexpressedinASHneurons(usingthesra-6promoter).
(I)Locomotionbehaviorofadulttransgenicwormswasanalyzedwithorwithoutcapsaicintreatment(5hours).Averagelocomotionvelocity(I)isplotted.
CapsaicintreatmentincreasedadultlocomotionvelocityintransgenicanimalsexpressingTRPV1inASHneurons,butnotinwildtypecontrols.Thenumber
ofanimalsanalyzedisindicatedforeachgenotype.(J)Thepercentageofanimalsparalyzedon1mMaldicarbat80minwithorwithoutcapsaicintreatment
(2–3hourspretreatment)wereplottedfortheindicatedgenotypes.Thenumberoftrialsisindicatedforeachgenotype.Fulltimecoursesforaldicarb-induced
paralysisareshowninS2GFig.CapsaicintreatmentincreasedaldicarbsensitivityintransgenicanimalsexpressingTRPV1inASHneurons,butnotinwild
typecontrols.ErrorbarsindicateSEM.Valuesthatdiffersignificantlyareindicated(***,p<0.001;ns,notsignificant).
doi:10.1371/journal.pgen.1005359.g004
adults(Fig5G).TheseresultssuggestthatGLR-2AMPAreceptorsarespecificallyrequiredfor
thearousedlocomotionduringtheL4/Alethargusinnpr-1mutants.
GLR-2AMPAreceptorsactinAIAandDVAtomediatearousal
WhichsynaptictargetsofASHandtouchneuronsmediatelocomotionarousal?Toaddress
thisquestion,weidentifiedtheneuronsinwhichGLR-2functionisrequired.ArousedL4/A
locomotionrequiresGLR-2butnotGLR-1receptors;consequently,wereasonedthattherele-
vantneuronsarelikelytoexpressGLR-2butnotGLR-1.GLR-1andGLR-2areco-expressed
inmanyneurons;however,afewGLR-2-expressingneuronslackGLR-1,includingDVA(a
stretch-activatedneuron)andAIA(aninterneuronintheheadganglia)[25–27].TheL4/A
quiescencedefectwaspartiallyrestoredinglr-2;npr-1doublemutantsbytransgenesexpressing
GLR-2inDVAandAIAneurons,whereastransgenesexpressedintheventralcordinterneu-
rons(usingtheglr-1promoter)failedtorescue(Fig5Band5C).Transgenicexpressionof
GLR-2inDVAorAIAhadnoeffectonlethargusquiescenceinwildtypeworms(S3Sig).
TheseresultssuggestthatGLR-2AMPAreceptorsexpressedinAIAandDVAneuronsarouse
L4/Alocomotioninnpr-1mutants.DVAreceivesdirectsynapticinputfromthetouchneuron
PLMwhileAIAreceivesdirectinputfromASH[28].Thus,increasedtransmissionat
ASH-AIAandPLM-DVAsynapsescouldaccountforGLR-2’seffectsonlocomotionrate.
Becauseweonlyobservedpartialrescuebyglr-2transgenesexpressedinAIAandDVA,itis
likelytheGLR-2functionisrequiredinadditional(asyetunidentified)neurons.
HowdoAIAandDVAarouselocomotion?AIAneuronsprovidesynapticinputtoASK
andASI,bothofwhichexpressPDF-1[11,29].Thus,heightenedAIAactivitycouldarouse
locomotionbyenhancingPDF-1secretion.ToassessthelevelofPDF-1secretion,weanalyzed
PDF-1::YFPfluorescenceintheendolysosomalcompartmentofcoelomocytes,whicharespe-
cializedscavengercellsthatinternalizeproteinssecretedintothebodycavity[30,31].Inactivat-
ingGLR-2didnotalterPDF-1::YFPfluorescenceincoelomocytesinbothadultandL4/A
animals(Fig6).TheseresultssuggestthatthearousingeffectsofGLR-2arenotmediatedby
changesinPDFsecretion.DVAneuronsreceivedirectsynapticinputfromthePLMtouch
neurons[32],andsecreteNLP-12(aneuropeptidethataccelerateslocomotion)[33].Thus,
increasedDVAactivitycouldcontributetolocomotionarousalinnpr-1mutants.Threeresults
supportthisidea.First,PLMneuronsexhibitenhancedtouch-evokedcalciumresponsesin
adultnpr-1mutants(S6Fig).Thus,PLMneuronshaveincreasedsensoryacuityinnpr-1
mutants,similartotheeffectwepreviouslyshowedforALMneurons[11].Second,inducing
DVAcelldeath(withaCED-3transgene)significantlyreducednpr-1locomotionrateduring
L4/Alethargus(Fig5H–5J),butnotinadults(Fig5K).Third,DVAsecretionofNLP-12issig-
nificantlyincreasedinnpr-1mutants[33],indicatingincreasedDVAactivity.Theseresults
suggestthatPLMneuronsprovideenhancedexcitatoryinputtoDVAinnpr-1mutants,which
promotesarousedL4/Alocomotion.
PLOSGenetics|DOI:10.1371/journal.pgen.1005359 July8,2015 9/20
GlutamateandNeuropeptidesArouseC.elegansLocomotion
Fig5.GLR-2AMPAreceptorsarerequiredforthenpr-1lethargusdefect.LocomotionbehaviorofsinglewormsduringtheL4/Alethargus(A-EandH-J)
andinadults(F,K)wasanalyzedintheindicatedgenotypes.Instantaneouslocomotionvelocity(A,H),averagemotilefraction(B,D,andI),andaverage
locomotionvelocity(C,E,F,JandK)areplotted.(A-C)Thenpr-1locomotiondefectduringL4/Aletharguswassuppressedbymutationsinactivatingglr-2
AMPAreceptors,andpartiallyreinstatedbytransgenesexpressingGLR-2inAIA(gcy-28(d)promoter)andDVA(nlp-12promoter)neurons,butnotinVentral
CordInterneurons(V.C.I.,glr-1promoter)inglr-2;npr-1doublemutantsusingtheindicatedpromoters.(D-E)glr-1mutationshadnosuppressingeffect.(F)
glr-2mutationsdidnotblocktheincreasedlocomotioninnpr-1adults.(G)mEPSCswererecordedfrombodywallmusclesoftheadultwormsforthe
indicatedgenotypes.Summarydataareshown.(G)glr-2mutationsdidnotblocktheincreasedmEPSCrateinnpr-1adults.(H-K)Locomotionbehavior
duringtheL4/Alethargus(H-J)andinadults(K)ofsinglewormswhoseDVAneuronisablatedbytransgenicoverexpressionofCED-3inDVAneuron(nlp-
12promoter)wasanalyzedintheindicatedgenotypes.AnimalswereanalyzedbyfluorescencemicroscopyafterlocomotionrecordingstodetermineifDVA
wasablated.Thenpr-1locomotiondefectduringtheL4/Alethargus,butnotinadults,waspartiallysuppressedinthetransgenicanimalsinwhichDVA
wasablated(-DVA).Thenumberofanimalsanalyzedisindicatedforeachgenotype.ErrorbarsindicateSEM.Valuesthatdiffersignificantlyareindicated
(**,p<0.01;***,p<0.001;ns,notsignificant).
doi:10.1371/journal.pgen.1005359.g005
Discussion
Toinvestigatethecircuitmechanismsforarousal,weanalyzedthelocomotionofnpr-1mutants
inawake(adult)andquiescent(lethargus)states.Ourresultsleadtofiveconclusions.First,mul-
tipleclassesofsensoryneuronscontributetoarousal.Second,diminishedsensoryacuityisa
PLOSGenetics|DOI:10.1371/journal.pgen.1005359 July8,2015 10/20