Synchronized Bilateral Synaptic Inputs toDrosophila melanogasterNeuropeptidergic Rest/Arousal ...

13 Pages·2011·4.74 MB·English

TheJournalofNeuroscience,June1,2011•31(22):8181–8193•8181
Cellular/Molecular
Synchronized Bilateral Synaptic Inputs to Drosophila
melanogaster Neuropeptidergic Rest/Arousal Neurons
Ellenav.McCarthy,1YingWu,1TagidedeCarvalho,2ChristianBrandt,3GuanCao,1andMichaelN.Nitabach1
1DepartmentofCellularandMolecularPhysiology,DepartmentofGenetics,PrograminCellularNeuroscience,NeurodegenerationandRepair,YaleSchool
ofMedicine,NewHaven,Connecticut06520,2DepartmentofEmbryology,CarnegieInstitutionforScience,Baltimore,Maryland21218,and3Centerfor
SoundCommunication,InstituteofBiology,UniversityofSouthernDenmark,DK-5230OdenseM,Denmark
NeuropeptidePDF(pigment-dispersingfactor)-secretinglargeventrolateralneurons(lLNs)intheDrosophilabrainregulatedailypatternsof
v
restandarousal.Thesebilateralwake-promotingneuronsarelightresponsiveandintegrateinformationfromthecircadiansystem,sleep
circuits,andlightenvironment.Tobegintodissectthesynapticcircuitryofthecircadianneuralnetwork,weperformedsimultaneousdual
whole-cellpatch-clamprecordingsofpairsoflLNs.BothipsilateralandcontralateralpairsoflLNsexhibitsynchronousrhythmicmembrane
v v
activitywithaperiodicityof(cid:1)5–10s.ThisrhythmiclLN activityisblockedbyTTX,voltage-gatedsodiumblocker,or(cid:1)-bungarotoxin,nicotinic
v
acetylcholinereceptorantagonist,indicatingthatactionpotential-dependentcholinergicsynapticconnectionsarerequiredforrhythmiclLN
v
activity.Sinceinjectingcurrentintooneneuronofthepairhadnoeffectonthemembraneactivityoftheotherneuronofthepair,thissuggests
thatthesynchronyisattributabletobilateralinputsandnotcouplingbetweenthepairsoflLNs.Tofurtherelucidatethenatureofthesesynaptic
v
inputstolLNs,weblockedoractivatedavarietyofneurotransmitterreceptorsandmeasuredeffectsonnetworkactivityandionicconduc-
v
tances.ThesemeasurementsindicatethelLNspossessexcitatorynicotinicAChreceptors,inhibitoryionotropicGABA receptors,andinhibi-
v A
toryionotropicGluCl(glutamate-gatedchloride)receptors.Wedemonstratethatcholinergicinput,butnotGABAergicinput,isrequiredfor
synchronousmembraneactivity,whereasGABAcanmodulatefiringpatterns.WeconcludethatneuropeptidergiclLNsthatcontrolrestand
v
arousalreceivesynchronoussynapticinputsmediatedbyACh.
Introduction 2000;Harmaretal.,2002;Albusetal.,2005;Atonetal.,2005,
Drosophilamelanogasterfliesexhibitrobustdailyrhythmsofrest 2006; Schneider and Stengl, 2005; Maywood et al., 2006;
andactivity,consistingoftwocrepuscularboutsofactivitywith Mertensetal.,2007).Theflylargeventrolateralneuron(lLNv)
an afternoon siesta in between. This complex daily pattern of subsetofcircadianneuronssecretestheneuropeptidepigment-
activity is generated coordinately by (1) the circadian rhythm dispersing factor (PDF) and their electrical activity has been
control circuit, (2) a homeostatic process regulating sleep, and shown to be directly light-responsive and is modulated by a
(3)lightinput(CirelliandBushey,2008;DubruilleandEmery, blue light-activated photopigment called cryptochrome (CRY)
2008;NitabachandTaghert,2008). (Sheebaetal.,2008a).Theseneuronsarewake-promotingand
TheDrosophilaneuralcircadiancontrolsystemiscomprised criticalfortheregulationofarousalandsleeppatterns(Collinset
of(cid:1)150clockneurons(Rennetal.,1999;Kanekoetal.,2000; al., 2005; Helfrich-Fo¨rster et al., 2007; Parisky et al., 2008;
Blanchardonetal.,2001;Helfrich-Fo¨rster,2004,2005;Nitabach Shangetal.,2008;Sheebaetal.,2008b).Thefunctionalsignals
andTaghert,2008).Intercellularcommunicationamongtime- from these neurons to downstream targets include activity-
keeping neurons via neuropeptide signaling and classical neu- modulatedPDFsecretion(Nitabachetal.,2002,2006;Wuet
rotransmission is essential for circadian rhythmicity in both al.,2008a,b).Thesefunctionaloutputsaremodulatedbythe
insects and mammals (Wagner et al., 1997; Liu and Reppert, intrinsic circadian timekeeping mechanism (Cao and Nit-
abach,2008),directactivationbylight(Sheebaetal.,2008a),
and by synaptic inputs, the nature of which are mostly un-
ReceivedApril20,2010;revisedApril13,2011;acceptedApril14,2011.
known, but likely include GABAergic input (Parisky et al.,
Authorcontributions:E.v.M.,Y.W.,G.C.,andM.N.N.designedresearch;E.v.M.,Y.W.,T.d.,C.B.,andG.C.per-
formedresearch;E.v.M.andY.W.analyzeddata;E.v.M.,Y.W.,andM.N.N.wrotethepaper. 2008;Chungetal.,2009).
ThisworkwassupportedinpartbyNINDS–NIHGrantsR01NS056443,R01NS055035,andR21NS058330(labo- Tobegintounravelthesynapticcircuitryoftheflycircadian
ratoryofM.N.N.).Y.W.wassupportedbyNINDS–NIHRuthL.KirschsteinNationalResearchServiceAward(NRSA) neural network, we used whole-cell patch-clamp physiology in
PostdoctoralFellowshipF32NS055527.E.v.M.wassupportedbyNationalInstituteofGeneralMedicalSciences–NIH
intact whole-brain explants. Simultaneous dual-cell recordings
RuthL.KirschsteinNRSAPostdoctoralFellowshipF32GM093344.T.d.andC.B.weresupportedinpartbyNational
frompairsoflLN s,regardlessofwhethertheywereinipsilateral
InstituteofMentalHealthGrantR25MH059472whilestudentsintheNeuralSystemsandBehaviorsummercourse v
attheMarineBiologicalLaboratory(WoodsHole,MA). or contralateral hemispheres of the brain, revealed highly syn-
CorrespondenceshouldbeaddressedtoMichaelN.Nitabach,DepartmentofCellularandMolecularPhysiology, chronousrhythmicmembraneactivity.RhythmiclLN activityis
v
DepartmentofGenetics,ProgramforCellularNeuroscience,NeurodegenerationandRepair,YaleSchoolofMedicine,
abolished by treating the preparation with either tetrodotoxin
333CedarStreet,NewHaven,CT06520.E-mail:[email protected].
(TTX),whichblocksactionpotentials,or(cid:1)-bungarotoxinorcu-
DOI:10.1523/JNEUROSCI.2017-10.2011
Copyright©2011theauthors 0270-6474/11/318181-13$15.00/0 rare,whichblocksnicotinicacetylcholinereceptors.Thesedata
8182•J.Neurosci.,June1,2011•31(22):8181–8193 McCarthyetal.•SynapticInputstoLargePDFNeurons
suggestthatcholinergicsynapticcommunicationisrequiredfor Dataacquisitionandanalysis.SignalsweremeasuredusingaMulti-
lLN rhythmicmembraneactivity. clamp 700B (Molecular Devices) and a Digidata 1440A (Molecular
v
To identify the nature of synaptic inputs to lLN s, we used Devices).
v
pharmacologicalmethodstoinhibitoractivateavarietyofneu- Thedegreeofsynchronyofthepairrecordingswasdeterminedby
filtering100sofcurrentclampdatausingalow-passGuassianfilter,and
rotransmitter systems and measured the effect on lLN mem-
v then running cross-correlation analysis on the pair of recordings
brane activity and ionic conductances. lLN s receive excitatory
v (Clampfit).Thepeakcorrelationvalueforeachpairwasdetermined.
inputvianicotinicacetylcholinereceptors(nAChRs)andinhib-
AutocorrelationwasusedtoanalyzetherhythmicRMPoscillationsin
itoryinputviabothGABAAreceptorsandglutamate-gatedchlo- 201Y(cid:4)/lLNvpairs.Again,100sofcurrent-clampdatawasfilteredusing
ridechannels(GluCls).CholinergicinputsarerequiredforlLNv alow-passGuassianfilter,andthenautocorrelationwasrun(Clampfit).
synchrony,whereasGABAergicinputisnotrequiredtomaintain Pairsexhibitingstrongburstfiringwereusedforstatisticalanalysis.
thissynchrony,butlikelyplaysamodulatoryroleinlLN mem- Theamplitudesofneurotransmitter-inducedcurrentsweremeasured
v
braneactivity. inClampfit,whichispartofthepClamp10softwarepackage.Thesig-
nificancetestswereperformedusingANOVAwithTukey–Kramermul-
MaterialsandMethods tiplecomparisonsonthedifferenceofthecross-correlationsofpaired
Clockneuronelectrophysiology recordingsandreversalpotentialsforglutamate-gatedcurrentindiffer-
AdultDrosophilawhole-brainexplantpreparation.Fliesweremaintained ent[Cl(cid:3)]osolutions.
at25°Cina12hlight/dark(LD)cycle.pdf-gal4;UAS-DsRedIIflylines
Drosophilabrainimmunostaining
wereusedasdescribedpreviously(BrandandPerrimon,1993;Rennet
Brainsfrompdf-Gal4/201Y-Gal4;UAS-dsREDfliesweredissected,fixed
al.,1999;Wuetal.,2008a).Fortheexperimentslookingatpairedrecord-
with4%paraformaldehyde,andthenstainedwithbothmouseanti-PDF
ings between lLNs and non-LNs, pdf-Gal4/201Y-Gal4;UAS-dsRED
v v (1:50) (Developmental Studies Hybridoma Bank, University of Iowa,
flieswereusedandtheneuronswereidentifiedbybothfluorescenceand
Iowa City, IA) and rabbit anti-dsRED (1:1000) (Invitrogen) primary
anatomicalposition.Threeto7dposteclosionfemalefliesthatexpress
antibodies.PDFanddsREDwerevisualizedusingaCy2-conjugated
redfluorescentprotein,DsRed,solelyinventrallateralclockneurons
anti-mouseantibody(1:300)(JacksonImmunoResearch)andaCy3-
(LNs)werecollectedforelectrophysiologicalrecordings.Whole-cellre-
v conjugatedanti-rabbitantibody(JacksonImmunoResearch),respec-
cordingsonlargeLNv(lLNvs)offlybrainexplantswereperformedas tively. The images were collected with a 20(cid:5) objective of a Zeiss
describedpreviously(GuandO’Dowd,2006;CaoandNitabach,2008;
AxioExaminerZ1microscope,usingAxioVision4software.
Wuetal.,2008a),andallindividualrecordingsweredoneinlightphase
ofLDcycle.Allpairedrecordingswereperformedbetweenzeitgebertime Results
22(ZT22)andZT23.Briefly,theflybrainsweredissectedinexternal
lLN sreceivesynchronoussynapticinputs
recordingsolution,whichconsistedofthefollowing(inmM):101NaCl, v
To address whether lLN s that regulate patterns of rest and
3KCl,1CaCl ,4MgCl ,1.25NaH PO ,5glucose,20.7NaHCO ,pH v
2 2 2 4 3 arousalexhibitsynchronousfiring,ashasbeenobservedinneu-
7.2,withosmolarityof250mmol/kg.Thebrainwasplacedventralside
up,securedinarecordingchamberwithamammalianbrainslice“harp” ralcircuitsinthemushroombodiesregulatingmemory(Rosayet
holder,andwascontinuouslyperfusedwithexternalsolutionbubbled al.,2001),weperformedwhole-cellpatch-clampphysiologyon
with95%O /5%CO atroomtemperature(22°C).lLNswerevisualized pairs of lLN s simultaneously (Fig. 1A). All paired recordings
2 2 v v
byDsRedfluorescence,andsubsequently,theimmediateareasurround- wereperformedatZT22–ZT23,whichwaschosentomaximize
ingthelLNswasenzymaticallydigestedwithfocalapplicationofpro- thenumberofburstfiringneurons,asthelargestpercentageof
v
teaseXIV(2mg/ml;Sigma-Aldrich). burstfiringlLN soccursatthistime(Sheebaetal.,2008a).Pairs
v
Whole-cellpatch-clampelectrophysiology.Whole-cellrecordingswere
oflLN neuronsresidinginthesamehemisphereexhibitedsyn-
performedusingborosilicatestandardwallcapillaryglasspipettes(Sutter v
chronized rhythmic membrane activity, such that the depolar-
Instrument).Recordingpipetteswerefilledwithinternalsolutioncon-
ized and hyperpolarized phases occurred simultaneously. This
sistingofthefollowing(inmM):102potassiumgluconate,17NaCl,0.085
synchronywasexhibitedinallpairsfromthesamehemisphere
CaCl ,4Mg-ATP,0.5Na-GTP,0.94EGTA,and8.5HEPES,pH7.2,and
osmo2larityof235mmol/kg.Theresistanceoffilledpipetteswasbetween thatexhibitedstrongburstfiring[peakcorrelationof0.8395(cid:6)
8and12M(cid:2).Gigaohmsealswereachievedbeforebreakingintowhole- 0.059(SEM);n(cid:7)8],whereastwopairsoflLNvsthatweretoni-
cell configuration in voltage-clamp mode, followed by break-in to callyfiringdidnotshowrhythmicmembraneactivity,andthere-
whole-cell configuration while in voltage-clamp mode. To confirm foresynchronycouldnotbedetected(Fig.1E).Wenextrecorded
maintenanceofagoodsealandabsenceofdamagetothecell,a40mV from pairs of lLN s from contralateral hemispheres (Fig. 1B).
v
hyperpolarizing pulse was imposed on each cell while in whole-cell Interestingly,allofthepairsofburstingneuronsalsoexhibited
voltage-clampmodefromaholdingpotentialof(cid:3)80mV.Onlyifthe
tight synchrony of electrical activity [peak correlation of
resultinginwardleakcurrentwaslessthan(cid:3)100pAwasthatcellusedfor 0.9112(cid:6)0.044(SEM);n(cid:7)5],whereasthreecontralateralpairs
subsequentcurrent-clampmeasurementsofrestingmembranepotential
that exhibited a mixture of tonic and burst firing showed de-
(RMP),actionpotential(AP)firingrate,andmembraneresistance.RMP
creasedsynchrony(Fig.1E).Therecordingsthatweperformed
wasdeterminedafterstabilizationofthemembranepotentialafterthe
transitionfromvoltage-clamptocurrent-clampmode,andforcellswith suggest that each member of the pair can switch firing modes,
oscillatingmembranepotentialwasdefinedatthetroughoftheoscilla- from burst to tonic and vice versa, independently, but exhibit
tion.APfiringratewascomputedoverthe5minperiodafterthetransi- synchronyonlyintheburstfiringmode.Ithasbeensuggested
tion from voltage-clamp to current-clamp configuration. Burst rate thatthismodulationbetweenfiringstatesmayberegulatedby
firingwasdefinedasthenumberofburstsoccurringinthelast60softhe voltage-gatedcalciumchannels(Sheebaetal.,2008a).
5minperiodafterthetransitionfromvoltage-clamptocurrent-clamp Toinvestigatewhethertherobustsynchronythatweobserved
configuration,dividedby60s.Membraneresistancewasmeasuredby
betweenlLN srepresentedageneralsynchronyofallneuronsin
injecting (cid:3)20 pA current in current-clamp recording mode. Voltage v
thewhole-brainpreparation[analogoustoepileptiformactivity
clampwasthenusedtorecordneurotransmitter-inducedcurrentson
lLNvsheldatdifferentholdingpotentialsinthepresenceof2(cid:2)MTTX sseyennchinromnoaumsmneauliraonnbarlaaicntisvliitcye]so(rFwishahetnh,e2r0i0t5h)adorsoomtheesrpfeocrimficsiotyf
(Sigma-Aldrich),whichinhibitsactionpotentialfiringofneuronsand
communication of neural network. Glutamate-induced currents were toneuronsassociatedwiththecircadianandarousalcircuits,we
alsorecordedinexternalsolutionwithreduced[Cl(cid:3)]obyreplacingNaCl performed paired recordings between lLNv and a stereotyped
withequalmolarofNa-gluconate. neuronthatispositiveforthewellstudieddriver201Y-Gal4and
McCarthyetal.•SynapticInputstoLargePDFNeurons J.Neurosci.,June1,2011•31(22):8181–8193•8183
A Ipsilateral lLNvs E1.0 * A -20 pA C 20 pA
lLNv #1
lLNv #1 n0.8 lLNv #1
o
-60mV elati0.6
orr
lLNv #2 Cross-C00..24 -60mV -60lmLNVv #2
-60mV lLNv #2
0.0
20mV Ipsilateral Contralateral 201Y+/lLNv
2 Sec. n=10 (8) n=8 (6) n=5 -60mV
B F 20mV
Contralateral lLNvs -60mV
20mV 200 ms
lLNv #1 1.0 2 0lL1NYv+
4 Sec.
n
atio0.5 B 20 pA D
lL-6N0vm #V2 correl0.0 lLNv #1 30 Injected Cell
o
Aut-0.5 ntial 20 Paired Cell
C -60mV 20mV2 Sec. -1.00 0.5 Tim1e.0 Lag (s1).5 2.0 -60mlLVNv #2 mbrane Pote-11000
201Y+ Neuron/lLNv Pair 1.0 2 lL0N1Yv+ Me-20
non-lLNv n
-50mV orrelation 00..05 -60mV 20mV Change i--4300
lLNv utoc 4 Sec. -50 -20 0 20
A-0.5 Current Injection (pA)
-50mV -1.0 Figure2. SynchronousmembraneactivityoflLN isattributabletosynchronizedsynaptic
20mV 0 0.5 1.0 1.5 2.0 2.5 v
input,nottocouplingbetweenthetwoneurons.A,Inthispairedrecording,thisrepresentative
D 2 Sec. Time Lag (s) tracedepictsonelLN thatwasinjectedwith(cid:3)20pAfor30s(top),whereastheresponsewas
v
simultaneouslyrecordedinanotherlLN inthesamebrainhemisphere(secondpanelfromtop).
d v
Inthisexample,thelLN thatwasinjectedwithnegativecurrenthyperpolarized,whereasthe
v
otherlLN wasunaffected(n(cid:7)6).Synchronyofrhythmicmembraneactivitybetweenthetwo
v
lLNswasunaffected.AllpairedrecordingswereperformedbetweenZT22andZT23.B,Simi-
v
larly,when20pAwasinjectedfor30sintoonelLN (bottom),thesecondneuronmaintainsits
v
baselinerestingmembranepotentialandisnotaffectedbythedepolarizationandactionpo-
tentialsinducedinthefirstlLN (n(cid:7)6).C,Zoominginonthefirst1softhedepolarizingcurrent
v
injectionintothefirstlLN,itisclearthatthereisnopostsynapticresponseofthesecondlLN to
v v
theactionpotentialfiringinthefirst.D,Quantificationoftheeffectofcurrentinjectiononboth
neuronsduringpairedrecordings,describedabove(n(cid:7)6).Theinjectedneurons(black)exhibit
v clearchangesinmembranepotentialasaresultofbeinginjectedwitheitherpositiveornega-
tivecurrent,whereaseachpairedneuronthatisconcurrentlyrecordedbutnotinjected(gray)
doesnotshowanychangeinmembranepotential.
Figure1. BilaterallysynchronousmembraneactivityoflLN rest/arousalneurons.A,B,
v
Representativesimultaneouswhole-cellcurrent-clamprecordingsoftwolLNvs,eitherfrom islocateddorsaltothelLN s(n(cid:7)5)(Fig.1C,D).All201Y(cid:4)/lLN
ipsilateral(n(cid:7)10)(A)orcontralateral(n(cid:7)8)(B)hemispheresinsitu.C,Simultaneous v v
pairsexhibitedonepeakofsynchronizedmembraneactivity,but
recordingshowsthepartiallysynchronizedfiringpatternsofonelLN andone201Y(cid:4)neuron.
v the 201Y(cid:4) neurons all demonstrated an additional peak of
Theseneuronsexhibitpartialsynchronizedmembraneactivity,sharingacommoninput,but
alsoreceivinguniqueinput(n(cid:7)5).AllpairedrecordingswereperformedbetweenZT22and rhythmicmembraneactivityofadifferentphase,notseeninthe
ZT23.D,Drosophilabrainat20(cid:5)magnificationstainedfortheneuropeptidePDFingreenand lLNvs(Fig.1C,F).Inaddition,thedegreeofsynchronythatwesee
fordsRED(expressedusingbothpdf-Gal4and201Y-Gal4,awellcharacterizedneuronaldriver). betweenthesepairsof201Y(cid:4)andlLN neuronsissignificantly
v
Thewhitearrowsindicatethelocationandidentityoftheneuronpairusedfor201Y(cid:4)/lLN differentfromtherobustsynchronyseenincontralaterallLN –
v v
pairedrecordings.The201Y(cid:4)neuronwasPDF(cid:3)andlocateddorsomedialtothelLNs.The lLN pairs(ANOVA,p(cid:7)0.02,p(cid:8)0.05)(Fig.1E).Thesedata
v v
dashedwhitelineindicatesthemidlineofthebrain,withdorsal(d)upandventral(v)down.E, showthat,whereasbothlLN sandthe201Y(cid:4)neuronsreceive
v
Cross-correlationanalysisofpairedrecordings.Allpairedrecordings,ipsilaterallLNv,contralat- somecommonrhythmicinputs,the201Y(cid:4)neuronsalsoreceive
eralLN,and201Y(cid:4)/lLN,werefilteredwithalowpassGuassianfilter,andthentheirdegreeof
v v uniqueinputsandarelesssynchronizedwithlLN sthanlLN s
synchronywasdeterminedbycross-correlation.Thecirclesrepresentthecorrelationalvalueof v v
aretoeachother.
eachpair;thegraycirclesindicatepairsexhibitingstrongburstfiring,whereastheunfilled
Todeterminewhetherthishighlysynchronizedelectricalac-
circlesrepresentpairsexhibitingdifferingdegreesoftonicfiring.Theblacksquaresshowthe
meandegreeofsynchronyofalltheburstingneuronsforeachconditionwiththeerrorbars tivitybetweenpairsoflLNvneuronsisaresultofsynchronized
representingtheSEM.Thenareindicated,withthenumberofburstingpairsincludedinthe synapticinputsor,rather,becauseofdirectcommunicationbe-
meaninparentheses.Thedegreeofsynchronyofthesegroupsweresignificantlydifferentfrom tweenlLN s,weexaminedtheeffectofsilencingorexcitingone
v
eachother(ANOVA,p(cid:7)0.02).Anasteriskdenotesstatisticalsignificance.F,Autocorrelation memberofapaironthemembraneactivityoftheother.Todo
analysisoftworepresentativepairsof201Y(cid:4)(black)andlLN (red)neuronsshowthatthey this,weachievedpairedrecordingsfromtwolLN sfromipsilat-
shareonecommonsynchronizedpeakofmembraneactivityvbutthatthe201Y(cid:4)neurons eral(n(cid:7)5)orcontralateral(n(cid:7)4)hemispheresv,theninjected
exhibitasecondpeakofmembraneactivitythatisnotsharedbythelLNs.
v negativeorpositivecurrentintooneofthepairtoinducedeither
8184•J.Neurosci.,June1,2011•31(22):8181–8193 McCarthyetal.•SynapticInputstoLargePDFNeurons
silencing or activation, respectively, and A
Ach Ach
observedthepatternoffiringintheother
memberofthepair.Injectionofnegative
current into lLN 1 hyperpolarized the
v
neuronandsuppressedmuchofitsaction -60 mV
10 mV
potential firing; the membrane potential B 20 sec
Nicotine
of this neuron continued to oscillate
aroundthishyperpolarizedbaseline(Fig.
2A,toptrace),indicatingthatthisneuron
was still receiving rhythmic synaptic in-
-70 mV Wash
puts. When we simultaneously observed
theelectricalactivityoflLN 2,itexhibited
v
unchanged membrane activity, continu-
ing to fire action potentials, in the same -70 mV Wash
wayasbeforethecurrentinjectionintoits
counterpart (Fig. 2A, second trace from
10 mV
top).ThesetwolLN sstillexhibitedsyn- -70 mV 20 sec
v
chronized rhythmic membrane activity. C Curare
Weobservednoeffectonthemembrane
activityofonelLN byinjectingnegative
v
currentintoanotherinallpairsexamined
(n(cid:7)9;5ipsilateralpairs,4contralateral -50 mV Wash
pairs). Similarly, when one lLN was in-
v
jected with positive current, the neuron
wastriggeredtofireaburstofactionpo- -50 mV 10 mV
20 sec
tentials,whereasitspairstayedataresting D α-BuTX
phase before both neurons receive syn-
chronizedsynapticinputs(Fig.2B).De-
spite this positive current injection into
-60 mV
lLN 1,lLN 2continuedtoreceivesynap-
v v
tic input, as evidenced by its exhibiting
postsynapticpotentials(PSPs).Thisinput
-60 mV 10 mV
wasnotfromitspairedlLN sincetheac- 20 sec
v
tionpotentialsfiredinlLNv1didnotcor- E 0 * * Baseline
p5r2eCaispi)prs.soiWlinandtjeeertcoaotbleptsdheawreivriPset,hdS4PpscsoiomssnieittleiravnarelaircnteuesrrlurLaleNltnspvtaw(i2nrits(h(cid:7))F.iaI9gnll;. Memrban eotPetnial (m)V------654321000000 ns Drugns
allcases,currentinjectionintoonelLNv -70 ACh Nicotine Curare a-BuTx
causedachangeinmembranepotentialin
Figure3. ModulationoflLN membraneactivitybynAChRagonistsandantagonists.A–D,Representativewhole-cellcurrent-
thatneuron,butnotinthelLN thatwas v
simultaneouslybeingrecordedfvrom(Fig. clamprecordingsofsinglelLNvsinsitu.A,ThisrecordingshowsoneexampleofanlLNvtreatedtwotimesconsecutivelywith1mM
AChfor5s.AChincreasedmembraneactivityofthelLN,depolarizingmembranepotentialandincreasingfiringrate.Thiseffect
2D).ThissuggeststhatlLNvsreceivesyn- wasreversibleandreplicable(n(cid:7)4).B,OneexamplevoflLN current-clamprecordingshowingtheeffectofthenAChRagonist
chronous synaptic inputs from neuron v
populationsdistinctfromthelLNvsthem- niniccoretainsee.dT(hne(cid:7)lLN8v)w.Aasfteexrptohseeedntdoo2fmnMicontiicnoetinexepfoosru1r0e,st.hMeeRmMbPraannedpAoPtefnirtiinalgarnadteacdteiocrneapsoetsensltoiawllfyirianngdrathteewneeurerodnraemxhaitbicitasllay
selves.Anadditionalpossibilityisthatsi- refractoryphase.Thebottomandmiddlepanelsshowtherestofthewashoutofthedrug,whichwasonlypartiallyreversiblein
lencing one lLNv in the context of the sevenofeightneurons.C,D,ThesearerepresentativerecordingsoflLNvstreatedwithnAChRantagonistscurare(200(cid:2)M)(n(cid:7)6)
network of eight lLNvs is not sufficient (C)or(cid:1)-bungarotoxin(0.5(cid:2)M)(D).Theinhibitoryeffectsofcurarearereversible,whereastheeffectsof(cid:1)-bungarotoxinwere
perturbation to disrupt the rhythmic irreversible,notreversingafter(cid:9)30minofwashout(datanotshown).E,Quantificationofchangesinmembranepotential
membraneactivityofanotherlLN ,even becauseofdrugtreatment.Theblackbarsindicatethemeanrestingmembranepotentialoftheneuronsbeforedrugtreatment,
v
if these neurons are synaptically con- whereasthegraybarsshowtheRMPofthecellafterpharmacologicalmanipulation.TheerrorbarsrepresenttheSEM.BothACh
nected.Wedonotbelievethisisthecase, andnicotineinducedsignificantchangesinRMP(p(cid:7)0.0016andp(cid:7)0.0007,respectively),whereascurareand(cid:1)-bungarotoxin
however,sinceseveringtheposteriorop- didnot.N(cid:9)5foreachtreatment.*,Statisticallysignificantdifference;ns,notsignificant.
tictract,amanipulationthatblockscom-
municationfromalllLN sinthecontralateralhemisphere,had 2000),wereasonedthatcholinergicinputwaslikelyamajorin-
v
no effect on synchronous membrane activity of contralateral fluenceonlLN membraneactivity.Totestthishypothesis,we
v
pairsoflLN s(datanotshown). examinedtheparticipationofcholinergicsignalsinlLN mem-
v v
braneactivitybyperformingwhole-cellpatch-clamprecordings
Nicotinicacetylcholinereceptorsmediateexcitatoryinputs onsinglelLN sincurrent-clampmode,withbathapplicationof
v
tolLN s agonistsorantagonistsofacetylcholinereceptors.Therepresen-
v
Sinceacetylcholineisthemostprevalentexcitatoryfastsynaptic tative lLN in Figure 3A exhibits oscillations in RMP between
v
neurotransmitterintheinsectCNS(Bossyetal.,1988;Schusteret (cid:3)55and(cid:3)40mVwithaburstofsixtoeightactionpotentials
al.,1993;YasuyamaandSalvaterra,1999;LittletonandGanetzky, duringthedepolarizedphase.Whenthepreparationwasbath-
McCarthyetal.•SynapticInputstoLargePDFNeurons J.Neurosci.,June1,2011•31(22):8181–8193•8185
treatedwith1mMacetylcholine(ACh),themembranedramati-
A
callydepolarizedandexperiencedaburstofAPs,lastingtensof
seconds(Fig.3A,E).Afterreturningtonormalsalinebath,themem-
brane gradually repolarized and membrane activity returned to
baseline.Thiseffectonmembranepotentialwasrepeatableandsig-
-50 mV
nificantnotonlyinthissameneuron,butalsoinotherlLNvsin 10 mV
independentwhole-brainexplants(p(cid:7)0.004;n(cid:7)4). 5 sec
HP -80 mV
TwodifferenttypesofAChreceptorshavebeenidentifiedin
the Drosophila nervous system, ionotropic and metabotropic
(Bossyetal.,1988;Schusteretal.,1991;Blakeetal.,1993).To
specificallytesttheeffectofionotropicAChreceptorsonmem- 50 pA
braneactivityoflLN s,webath-appliedaspecificionotropicag- 5 sec
v
onist,nicotine,andrecordedtheresultingresponseincurrent-
clamp mode. When 2 (cid:2)M nicotine was added to the external 2 sec
solution, lLN s underwent dramatic depolarization of their
v
membranepotential,which,afterasmallamountofrepolariza- 20 pA 0.1 sec
tion,wasfollowedbyaburstofAPfiringandthenaplateau(Fig.
B
3B,E). The depolarized plateau ranged from (cid:3)20 to (cid:3)6 mV TTX
(mean,(cid:3)10.97(cid:6)1.31mV)withadurationof14to200s(n(cid:7)8),
continuinglongaftertheremovalofnicotinefromthebathso-
lution.Themembranepotentialduringnicotinetreatmentsig-
nificantlyelevatedoverbaseline(p(cid:8)0.00001).Theprolonged
-60 mV 10 mV
natureofthiseffectcouldpossiblybeattributabletotheveryhigh
10 sec
affinityforthisagonisttoitsreceptor.Thedepolarizedstategrad- C
HP -80 mV
uallygavewaytorepolarizationoftheRMPandadecreaseinAP
firing.Interestingly,theseneuronsexhibitedarefractoryphase
beforereturningtorhythmicAPfiring.Thiseffectwasonlypar- -50 mV 2 sec
tiallyreversible,asonlyoneneuronofeighttestedreturnedback
10 mV
to its firing rate before nicotine application. This complex re- 20 pA 0.1 sec
sponseoflLN stonicotineraisesthepossibilitythatthisnAChR
v
D E
agonistnotonlyexciteslLN membraneactivitybutalsoexcites Ach Nicotine
v
inhibitorysynapticinputstolLN s,assuggestedbytherefractory HP (mV) HP (mV)
v
phaseafterrepolarization.TotesttheeffectofmetabotropicACh
receptorsonlLN membraneactivity,muscarine,ametabotropic 10 10
v
AChRagonist,wasappliedtotheexternalsolutionandthere-
sponse was recorded (supplemental Fig. 1, available at www. 0 0
jneurosci.orgassupplementalmaterial).Thistreatmentinduced
nosignificantdepolarizationorchangeinintervalbetweenbursts -10 -10
ofAPsbutdidresultinanincreaseinAPnumber(meanbaseline 50 sec
APfiringrate,1.925(cid:6)0.248Hz;meanmuscarineAPfiringrate, -20 -20
4.1(cid:6)0.414Hz;p(cid:8)0.004),whichwasalessdramaticresponse
comparedwiththatelicitedbytheionotropicagonist,nicotine.
-30 -40
Given that the nicotinic agonist induced such a strong re-
40 pA
sponsefromlLN s,weexaminedtheeffectofnAChRantagonists, 20 pA
v 50 sec
curareand(cid:1)-bungarotoxin((cid:1)-BuTX)onlLN membraneactiv- 50 sec
v
ity.Bathapplicationof200(cid:2)McurareeliminatedAPfiringal-
Figure4. nAChRcurrentsoflLNs.A,RepresentativetraceoflLN spontaneousactivityin
mostimmediately,butdidnotchangemembranepotential,and v v
current-clampmode(A,top)orvoltage-clampmodewithaholdingpotentialof(cid:3)80mV(A,
wascompletelyreversibleonwashoutofthedruginfourofsix
middle).ThebottomtwotracesinAareenlargementsofpartsoftherestphasethatareboxed
neuronstested(oneneuronshowedonlypartialrecovery)(Fig.
inthepaneldirectlyaboveeachone.B,Representativecurrent-clamprecordingoflLN treated
3C,E).Similarly,(cid:1)-BuTXinhibitedmembraneactivityoflLN s, v
v withTTX.TTXabolishedactionpotentialsandrhythmicmembraneactivity.C,Representative
butdidnotchangemembranepotential(n(cid:7)14)(Fig.3D,E), current-clamp(left)andvoltage-clamp(right)recordingsfromasinglelLN treatedwithTTX
v
althoughthekineticsofitseffectwereslowerthanthatofcurare, andmagnifiedviewsoftheregionsboxedineachtoppanel,respectively.Miniaturepostsyn-
possibly because of the fact that curare is a small molecule, as apticpotentials(left)andcurrents(right)arevisible.D,E,Voltage-clamprecordingsofACh-(D)
opposed to a peptide like (cid:1)-BuTX, and therefore was able to ornicotine-induced(E)currentsinindividuallLNsatarangeofholdingpotentials(10,0,(cid:3)10,
v
diffuse into (and out of) the whole-brain preparation more (cid:3)20,or(cid:3)30mV).ACh-andnicotine-inducedcurrentsreversedaround(cid:3)10mV.
quickly.IntherepresentativetraceshowninFigure3D,onaddi-
tion of 0.5 (cid:2)M (cid:1)-BuTX, the lLNv exhibited progressively de- neuronsandnodetectablerecoveryinsix.Importantly,curareor
creased firing with an (cid:1)5 mV oscillation in RMP, which (cid:1)-BuTX blockade of nAChR each resulted in loss of rhythmic
transitionedtocompleteabolitionofAPfiringandarelatively oscillationinmembranepotential,indicatingthattheseoscilla-
stableRMP(Fig.3D).Ofthe14neuronstested,(cid:1)-BuTXcaused tionsarenotintrinsicpacemakerpotentials,butratherareim-
completecessationofAPfiringin10lLN sandpartialblockof posedbysynchronizedsynapticinputs.
v
APfiringintheotherfour.Theinhibitoryeffectofthistoxinwas TheinhibitionofactionpotentialfiringinlLN sbycurareand
v
not reversible, as washout led to only partial recovery in eight (cid:1)-BuTXanddramaticdepolarizationinducedbynicotinesug-
8186•J.Neurosci.,June1,2011•31(22):8181–8193 McCarthyetal.•SynapticInputstoLargePDFNeurons
A B C
GABA *
0 5
-10 Hz) HP (mV) GABA
-70 mV GABA eMmrban ePotentila (mV)------765432000000 ns ActionP otnetial iFring Rate (1234 ---344005
-80 0
- + - +
GABA GABA
-50
-70 mV 20 mV
20 sec -70
20 pA
50 sec
D E
Picrotoxin
*
0 8 * 1.0
-70 mV Mmebraen Potetinl a(m)V------654321000000 ns n Potneati ilFring Rat(e zH)234567 u rsBtrFeqeunc y(Hz)0000....2468
-70 Actio1
-80 0 0.0
- + - + - +
Picrotoxin Picrotoxin Picrotoxin
-70 mV
20 mV
20 sec
2.5 sec
Figure5. GABAergicmodulationoflLNvmembraneactivity.A,Representativecurrent-clamprecordingoflLNvresponsestobath-appliedGABA(1mM)(n(cid:7)5).IntwotrialsonthesamelLNv,GABAreversibly
abolishedactionpotentialsandrhythmicmembraneactivity.B,QuantificationoftheeffectofGABAonRMPandactionpotentialfiringoflLNs.TheblackbarsrepresentthemeanRMP(left)oractionpotential
v
firingrate(right),beforeandafterGABAtreatment.TheerrorsbarsequaltheSEM.TreatmentoflLNswithGABAcausesasignificantdecreaseinAPfiring(p(cid:7)0.029)whilecausingnochangeinRMP(p(cid:9)0.05).
v
*,Statisticallysignificantdifference;ns,notsignificant.C,Voltage-clampmeasurementofGABA(1mM)-inducedcurrentsatarangeofholdingpotentials((cid:3)30,(cid:3)40,(cid:3)45,(cid:3)50,or(cid:3)70mV)inthepresence
ofTTX.GABA-inducedcurrentsreversedbetween(cid:3)45and(cid:3)50mV,nearthecalculatedequilibriumpotentialofCl(cid:3).D,Representativecurrent-clamprecordingoflLN treatedwiththechloridechannel
v
antagonistpicrotoxin(100(cid:2)M)(n(cid:7)9).Picrotoxincausesthetroughofrhythmicmembraneactivitytobemoredepolarizedandincreasesthenumberofactionpotentialsineachburst(top).Bottomtracesare
enlargementsofboxedregionsinthetoptrace.E,QuantificationoftheeffectofpicrotoxinonRMP,actionpotentialfiring,andburstfiringfrequencyoflLNs.TheblackbarsrepresentthemeanRMP(left),action
v
potentialfiringrate(middle),orburstfiringfrequency(right)beforeandafterpicrotoxintreatment.TheerrorsbarsequaltheSEM.TreatmentoflLNswithpicrotoxincausesasignificantincreaseinAPfiring(p(cid:7)
v
0.0025)andinburstfiringfrequency(p(cid:7)0.0008)whilecausingnosignificantchangeinRMP(p(cid:9)0.05).*,Statisticallysignificantdifference;ns,notsignificant.
gestthatnAChRsmediateexcitatorysynapticinputtotheseneu- righttraces).ItisreasonabletoconcludethattheTTX-resistant
rons. However, this does not rule out the possibility that the small inward currents are miniature postsynaptic currents
observedeffectsarenotattributabletodirectcholinergicsynaptic (mPSCs),mediatedbyspontaneousvesiclereleasebypresynaptic
input into lLN s, but rather indirect effects from intermediary neuronswithinputintolLN s.Theamplitudeoftheseminiature
v v
neuronsinterposedbetweencholinergicneuronsandlLN s.To currentsissimilartothoseobservedinKenyoncellsintheDro-
v
directlyassessnAChR-mediatedcurrentsinlLN s,weusedvolt- sophilamushroombody(SuandO’Dowd,2003).Theeffectof
v
ageclamptomeasureACh-inducedcurrentsinthepresenceof TTXonlLN thatweobserveisconsistentwiththeeffectofTTX
v
bath-appliedTTX.TTXblocksvoltage-gatedsodiumchannelsin onlLN spreviouslyreported(Sheebaetal.,2008a),despitethe
v
allneuronsinthewhole-brainexplant,thusinhibitingAPfiring factthattheyuseda20-foldlowerdose.
inallneuronsandtherebypreventingallnonspontaneoussynap- In our experimental paradigm, we first established a robust
ticactivity.Innormalbathsolution,lLN sexhibitspontaneous recording,assessingthemembraneactivityofalLN incurrent-
v v
actionpotentialfiring,tonicorbursting,andoftenoscillationsin clampmode,andthenaddedTTXandmonitoredthelossofAPs,
RMP(Fig.4A,toptrace).Involtage-clampmodewithaholding asinFigure4B.WethenrecordedAChRagonist-inducedcur-
potentialof(cid:3)80mV,lLN sexhibitrhythmiclargeinwardcur- rentsataseriesofholdingpotentialsanddeterminedthereversal
v
rents of (cid:1)100 pA, corresponding to AP-dependent excitatory potentialforthesecurrentsbyassumingalinearrelationshipin
synapticinputs,andalsoshowedsmallinwardcurrents,ranging thisrangeofholdingpotentialsforeachindividualneuron.Inthe
inamplitudefrom2to10pA,duringtherestingphase(Fig.4B, representativerecordinginFigure4D,applicationof1mMACh
bottomtraces).WhenTTXwasaddedtothebathsolution,action inducedanoutwardcurrentatpotentialspositiveto(cid:3)10mVand
potential firing and rhythmic membrane activity were blocked aninwardcurrentatpotentialsnegativeto(cid:3)20mV(Fig.4D).
(Fig. 4B). However, small TTX-resistant transient depolariza- Afterthereversalpotentialwasdeterminedformultipleindivid-
tionscouldstillbeobservedincurrentclamp(Fig.4C,lefttraces) uallLN s(n(cid:7)7),thereversalpotentialofthecurrentwasdeter-
v
and corresponding inward currents in voltage clamp (Fig. 4C, mined by averaging the reversal potentials of each individual
McCarthyetal.•SynapticInputstoLargePDFNeurons J.Neurosci.,June1,2011•31(22):8181–8193•8187
A Glutamate a variety of factors, including kinetics of
wash-inoftheagonisttovariousregions
oftheextensivedendriticarborsofthelL-
N s,onandoffbindingkineticsofagonist
v
with the receptor, desensitization of the
-50 mV receptors, or the inability to hold the
wholesurfaceoftheneuronattheholding
potentialsetatthesoma.
GABA receptorsmediateinhibitory
-50 mV 20 mV A
synapticinputstolLN s
v
10 sec GABA is a major inhibitory neurotrans-
B C mitterininsectsandiswidelydistributed
Membrane Potential (mV)------6543210000000 * on PotentilaF iring Rtae (Hz) 12345 * RP (mV) ----1234 00000 114 [Cl-]o 63.5 [Cl-]o 17 [Cl-]o iAH2spwni0rorioet0otnhssh0nisee)sid.tntCeheSetcNeiintnarhGlSec.ea,ea(Ais1frnleBfL9rdd9NLAe7nNGAvf;scfALrrhhseeiB-atcnCstvAechleepohAotntb-owoCrsenreteoacndaannennisptssddhrtteauootGsnprwaiatntl,neen.c,xdeet11pot99zfrrle99keeixe15sys--st,;;
-70 Acti (Pariskyetal.,200v8;Chungetal.,2009),
-80 0 -50
- + - + wehypothesizedthatGABAmightbean
Glutamate Glutamate
importantinhibitoryinputintolLN s.To
D v
test this, we first examined the effect of
HP (mV) Glutamate/114[Cl-] Glutamate/63.5[Cl-] Glutamate17[Cl-]
o 5o0 pA o GABAapplicationonthemembraneac-
0
-10 20 tivity of lLN s in current-clamp mode.
v
When GABA (1 mM) was added to the
-40 -20 0 externalsolution,APfiringwasrapidly
blocked(n(cid:7)5),butthemembranepo-
-45 -30 -10 tentialwasnotsignificantlyaltered(p(cid:9)
0.05).Thiseffectwasreversibleandcould
be repeated in the same neuron (Fig.
-50 -40 -20 5A,B).ThisactivationofGABAreceptors
also resulted in a loss of oscillations in
-70 -60 -40 resting membrane potential in lLNvs.
30 pA 30 pA 20 pA Conversely, when picrotoxin, a GABA
A
50 sec 50 sec 50 sec receptor antagonist, was bath applied, it
increased firing rate (mean baseline AP
Figure6. GlutamatergicmodulationoflLNvmembraneactivity.Representativecurrent-clamprecordingoflLNvresponsesto firing, 1.7 (cid:6) 0.474 Hz; mean picrotoxin
bath-appliedglutamate(1mM).A,IntwotrialsonthesamelLNv,glutamate(1mM)reversiblyabolishedactionpotentialsandrhythmic APfiringrate,5.94(cid:6)1.09Hz;p(cid:7)0.0025)
membraneactivity(n(cid:7)5).B,QuantificationoftheeffectofglutamateonRMPandactionpotentialfiringoflLNs.Theblackbarsrepresent
v andalsoincreasedthefrequencyoffiring
themeanRMP(left)oractionpotentialfiringrate(right),beforeandafterglutamatetreatment.TheerrorsbarsequaltheSEM.Treatment
oflLNswithglutamatecausesasignificantincreaseinRMP(p(cid:7)0.0008)andaconcurrentdecreaseinAPfiring(p(cid:7)0.003).*,Statistically bursts (mean baseline burst frequency,
signifivcantdifference;ns,notsignificant.C,Voltage-clampmeasurementofglutamate(1mM)-inducedcurrentsinthepresenceofTTX. 0.346(cid:6)0.114Hz;meanpicrotoxinburst
Glutamate-inducedcurrentsreversedatholdingpotentialsthatvariedwith[Cl(cid:3)] andwerenearthecalculatedreversalpotentialsCl(cid:3)at frequency,0.9(cid:6)0.057;p(cid:7)0.0008).The
o
each[Cl(cid:3)].D,Quantificationoftheexperimentallyobservedreversalpotentialsofglutamate-inducedcurrentsatdifferent[Cl(cid:3)] (black picrotoxineffectwasalsoreversible.
o o
bars)versusthecalculatedreversalpotential.TheerrorbarsdepicttheSEM. To determine whether this response
could result from GABA-induced cur-
neuron(supplementalFig.2,availableatwww.jneurosci.orgas rentswithinlLN sthemselves,weperformedvoltage-clampre-
v
supplemental material). The reversal potential for the ACh- cordingsinthepresenceofTTX(asinFig.4above).Application
induced current was (cid:3)8.9 (cid:6) 1.4 mV (SEM). Similarly, we re- of GABA induced an outward current at holding potentials at
cordednicotine-inducedcurrentsinlLN s(representativetrace) (cid:3)45mVoraboveandaninwardcurrentatpotentialsof(cid:3)50mV
v
(Fig.4E)andsubsequentlydeterminedthereversalpotentialforthe orbelow(Fig.5C).Wedeterminedthereversalpotentialofthe
nicotine-inducedcurrenttobe(cid:3)14.9(cid:6)1.4mV(supplementalFig. GABA-inducedcurrentinlLN stobe(cid:3)48(cid:6)0.6mV(n(cid:7)5)
v
2, available at www.jneurosci.org as supplemental material). As (supplementalFig.2,availableatwww.jneurosci.orgassupple-
expected,theexperimentallymeasuredreversalpotentialsforACh- mentalmaterial),whichisnearthecalculatedequilibriumpoten-
andnicotine-inducedcurrentsarenearthepredictedreversalpoten- tialforCl(cid:3),(cid:3)48.1mV.Theseresultsstronglysuggestthatthe
tial for a nonselective monovalent cation channel. These data GABA receptor is present in lLN s and that these receptors
A v
confirmthatnAChRsarepresentinlLNsandmediateexcitatory mediate fast inhibitory synaptic inputs, which are important
v
synapticinput. for appropriate regulation of rest and arousal (Parisky et al.,
Both the ACh- and nicotine-induced currents displayed a 2008;Chungetal.,2009).Thesedataareconsistentwithprevious
slightlydelayedonsetinrelationtothebathapplicationofthe physiological recordings demonstrating GABA -mediated cur-
A
agonist and also exhibited repeatable complex time courses of rentsindissociatedPDF-positiveneuronsinculture(Chunget
activation.Theserepeatabletimecoursescouldbeattributableto al.,2009).
8188•J.Neurosci.,June1,2011•31(22):8181–8193 McCarthyetal.•SynapticInputstoLargePDFNeurons
Glutamate-gatedCl(cid:1)channelsmediate A
Acetylcholine
inhibitoryinputtolLN s
v lLNv #1
Glutamate is an important inhibitory
neurotransmitterininvertebratenervous
systems, as it gates the GluCl that is not
presentinmammalsorothervertebrates -50mV
lLNv #2
(Cullyetal.,1994,1996;Dentetal.,1997,
2000; Laughton et al., 1997; Vassilatis et
al.,1997;Cooketal.,2006).GluClcriti- 10 mV
-50mV 5 Sec
cally regulates patterns of locomotor Washout
lLNv #1
activity and pharynx function in Caeno-
rhabditiselegans(Dentetal.,1997;Cook
et al., 2006). Although this neurotrans-
mitterreceptorwasclonedinDrosophila -40mV
lLNv #2
byhomologywithitsC.eleganscounter-
part(Cullyetal.,1994)anditsphysiology
has been characterized in vitro (Cully et
al., 1996), we are unaware of any func- -40mV
10 mV
tionalroleforGluClthathasbeenidenti- B 10 Sec
fied in Drosophila in vivo. To test the lLNv #1 Curare
hypothesis that GluCl inhibits network
activity, we recorded from lLN s in
v
current-clamp mode and applied 1 mM -50mV
lLNv #2
glutamate in the bath. As predicted by
GluCl biophysical properties, glutamate
application immediately blocked AP fir-
ing,andstabilizedmembranepotentialat
10 mV
around(cid:3)40mV,whichwasreversedon -60mV 5 Sec
washout (Fig. 6A) (n (cid:7) 5). The mean Washout
lLNv #1
baseline resting membrane potential be-
fore addition of glutamate was (cid:3)57.0 (cid:6)
2.43mV,butdepolarizedto(cid:3)40.8(cid:6)1.97 -50mV
lLNv #2
mV after glutamate treatment (p (cid:7)
0.0008)(Fig.6B).
To determine whether this effect of
glutamate on lLNv membrane activity C -50mV 10 mV 10 Sec
couldbemediatedbydirectactivationof 1.0 1.0
pGelrufCorlmcheadnnvoellstaigne-lcLlNamvspthreemcosredlvinegs,swine elaiton0.8 leation0.8
the presence of bath-applied TTX to re- oCrr0.6 oCrr0.6
cord glutamate-induced currents of Crsos-0.4 Cross-0.4
lLN s.Glutamateinducesaninwardcur-
rentvatholdingpotentialsof(cid:3)45mVor 0.2 0.2
belowandanoutwardcurrentat(cid:3)40mV 0.0 Before During After 0.0 Before During After
orabove,asshowninarepresentativecell Acetylcholine Treatment Curare Treatment
inFigure6C.Afterrepeatingthisinmul-
Figure7. ActivationofAChreceptorsorinhibitionofcholinergicinputsdisruptslLN synchrony.A,Representative
triepvleerslaLlNpvoste(nnti(cid:7)alt8o),bwee(cid:3)d4e4t.e1rm(cid:6)in0e.6dmthVe swimasuhletadnoeuotu(sbwothtoolme-tcreallcceus)r.reAnllt-pcalairmedprreeccoorrddiinnggsswofetrweopeiprfsoilramteerdalblLeNtwvseterneaZtTe2d2wainthdAZTC2h3v(.1Bm,MR)e(pnre(cid:7)sen5)t,awtivheicshimwualsttahneen-
(supplemental Fig. 2, available at www. ouswhole-cellcurrent-clamprecordingsoftwoipsilaterallLNvstreatedwithcurare(200(cid:2)M)(n(cid:7)6),whichwasthen
jneurosci.org as supplemental material), washedout(bottomtraces).C,QuantificationofthecorrelationofmembraneactivitybetweenpairsoflLNsbefore,
v
which,similartotheGABA-inducedcur- during,andafterACh(left)orcurare(right)treatment.Eachpairthatwastreatedwiththeseagentsisshown(n(cid:7)5and
rent (Fig. 5), is close to the equilibrium n(cid:7)6,respectively).
potential of Cl(cid:3). To confirm that the
ionicbasisfortheglutamate-inducedcurrentinlLN sisCl(cid:3),we effectofglutamateonlLN firingrateisnotattributabletoGluCl
v v
recorded the glutamate-induced current in external solutions conductance, but rather attributable to activation of metabo-
withreduced[Cl(cid:3)] .Whenwereduced[Cl(cid:3)] ,thereversalpo- tropic glutamate receptors, whose downstream G-protein-
o o
tentialofthecurrentbecamesignificantlymoredepolarized(p(cid:8) dependentsignalingcausestheopeningofachlorideionchannel,
0.001,ANOVA)(Fig.6D;supplementalFig.2,availableatwww. resultinginacurrentthatreversesatthechloridereversalpoten-
jneurosci.orgassupplementalmaterial),andcloselytrackedthe tial. Although this is a formal possibility, we consider it more
predictedCl(cid:3)equilibriumpotentials.Together,thesedataindi- likelythatGluClisresponsibleforthecurrentweobserve,asthe
catethatCl(cid:3)istheprimaryionicbasisfortheglutamate-induced roleofmGluRonionicconductancehasbeenwidelyinvestigated
currentandthatGluClchannelsinlLN scontributetotheirsyn- intheliteratureandnosuchactivationofchloridechannelsby
v
apticinhibition.Onealternativepossibility,however,isthatthe mGluRsignalinghasbeenobserved.
McCarthyetal.•SynapticInputstoLargePDFNeurons J.Neurosci.,June1,2011•31(22):8181–8193•8189
A Glutamate Similarly, when lLNvs were treated
with glutamate, all synchronous mem-
lLNv #1
braneactivitybetweenlLN pairswaslost
v
(Fig.8)(n(cid:7)5).Inseveralcases,synchro-
nized burst firing returned on washout
(fouroffivepairs),butaswithcholinergic
-50mV agentwashout,thetimingofthereturnto
lLNv #2
synchronywasvariable,withtwopairsbe-
ginning burst firing simultaneously and
tworegainingrhythmicmembraneactiv-
ityindependently.
-50mV Incontrast,treatmentoftheseneurons
withpicrotoxin,whichinhibitedendoge-
10 mV
Washout 5 Sec nousGABAergicinputtotheseneurons,
didnotleadtolossofsynchronybetween
lLNv #1
thetwoneurons(Fig.9A,B)(n(cid:7)5).In
fact,twopairsofneuronsthatweretoni-
cally firing, and therefore not exhibiting
synchronous firing before picrotoxin
treatment,beganburstfiringandbecame
-50mV
lLNv #2 moresynchronizedwithpicrotoxintreat-
ment(Fig.9C).Thesedataareastarkcon-
trast to the effect we observed when we
inhibitedendogenouscholinergicsignal-
ingwithcurare.Inthecaseofcurare,in-
hibition of the endogenous cholinergic
input lead to complete abolition of syn-
-50mV
10 mV chronous membrane activity between
10 Sec lLN pairs.Becauseofthelackofaspecific
v
GluCl antagonist, we could not directly
B
testthenecessityofendogenousglutama-
1.0
tergicinputinlLN synchrony.Together,
n v
taio 0.8 these data suggest that endogenous cho-
le
rro 0.6 linergic input is essential for lLNv syn-
C
s- chronous membrane activity, whereas
Cros 0.4 GABAergic input is not required, but
0.2 canmodulatethefiringpropertiesofthe
neurons.
0.0
Before During After
Discussion
GlutamateTreatment
Through the use of simultaneous dual
Figure8. ActivationofglutamatereceptorssilencessynchronouslLNvfiring.A,Representativesimultaneouswhole-cell whole-cell patch-clamp recordings in
current-clamprecordingsoftwoipsilaterallLNvstreatedwithglutamate(1mM)(n(cid:7)5),whichwasthenwashedout(bottom Drosophila whole-brain explants, we ob-
traces).AllpairedrecordingswereperformedbetweenZT22andZT23.B,Quantificationofthecorrelationofmembraneactivity
servedsynchronousmembraneactivityin
betweenpairsoflLNsbefore,during,andglutamatetreatment.Eachpairthatwastreatedwiththeseagentsisshown(n(cid:7)5).
v lLN s mediated by bilateral synaptic in-
v
Cholinergicinputs,butnotGABAergicinputs,arerequired puts.PairsoflLN sfromeitherthesame
v
forlLN synchrony orcontralateralhemispheresexhibitedsynchronousrhythmsof
v
OurdatasuggestthatlLN sreceivecholinergic,GABAergic,and membraneactivity(Fig.1A,B).Ourdataindicatethatthisrobust
v
glutamatergicinputs,sowenextinvestigatedtheneurochemical synchronyisattributabletosynchronizednetworksynapticin-
basis for the robust synchronous synaptic inputs that we ob- puts,asopposedtodirectelectricalorsynapticcouplingbetween
servedinpairsoflLN neurons.Todothis,weperformedwhole- lLN s,asmanipulationofthemembraneactivityofoneneuron
v v
cell patch-clamp physiology on ipsilateral pairs of lLN s throughnegativeorpositivecurrentinjectiondidnotalterthe
v
simultaneously, then applied either ACh, curare, glutamate, or membraneactivityofitssynchronouspair(Fig.2).
picrotoxintothebathandobservedtheresponseofbothneu- Ithasbeenpreviouslyshownthatneuralcircuitsresponsible
rons.AllpairsoflLN streatedwithAChorcurare,toactivateor forgeneratingcircadianrhythmsandalsothoseneuralnetworks
v
inhibitcholinergicsynapticinput,showedadramaticdecreasein controllingrestandarousalexhibitsynchronousmembraneac-
synchronyduringtreatment(Fig.7)(n(cid:7)5andn(cid:7)6,respec- tivitybothinmammalsandininsects(Nitzetal.,2002;Schneider
tively).Onwashout,6of11pairsregainedsynchronousmem- andStengl,2005;Welshetal.,2010).Furthermore,ithasbeen
braneactivity(Fig.7C).Ofthepairsthatregainedsynchronyon shownthatneuropeptides,VIPandPDF,inmammalsandflies,
washout,fourregainedburstfiringsimultaneously.Inthecaseof respectively,andtheclassicalneurotransmitter,GABA,playcrit-
theothertwopairs,onecellregainedburstfiringfirst,whereas icalrolesinthissynchrony(InouyeandKawamura,1979;Welsh
the other fired tonically, but regained burst firing later in the etal.,1995;Wagneretal.,1997;LiuandReppert,2000;Shirakawa
washout. etal.,2000;Albusetal.,2005;Atonetal.,2005,2006;Schneider
8190•J.Neurosci.,June1,2011•31(22):8181–8193 McCarthyetal.•SynapticInputstoLargePDFNeurons
andStengl,2005).Furthermore,wefound
thatstereotypedneuronsthatwereposi-
tive for a well studied driver exhibited
varying degrees of synchrony with lLN
v
membrane activity (Fig. 1C,E). This is
consistentwithamodelinwhichcertain
neuronsreceivesomeofthesameinputs
aslLN sandsomeuniqueinputs.Similar
v
to our observations in Drosophila, neu-
rons in some mammalian brain regions
outside the suprachiasmatic nucleus
(SCN) exhibit synchronized membrane
activitieswithSCNneurons(Inouyeand
Kawamura,1979).Ourdatadonot,how-
ever,precludethepossibilitythatthesyn-
chronythatweobserveisattributableto
widespread epileptiform or other wide-
spreadsynchronousbrainactivitythatis
notspecifictolLN s.Wedonotfavorthis
v
explanation because we have observed
lLN pairs exhibiting varying degrees of
v
synchrony and also lLN pairs in which
v
one cell is burst firing while the other is
tonicallyfiring,asshowninourpairedre-
cordingpharmacologyexperiments.Even
ifitisthecasethatthesynchronizedactiv-
itythatweseeisattributabletosomesort
ofepileptiformorotherwidespreadsyn-
chronous activity, the nature of and
Figure9. InhibitionofGABAergicinputsdoesnotblocklLN synchronybutdoesmodulatelLN firingpattern.A,
mechanisms underlying this activity are v v
stillinformative,asitprovidesinsightinto R(nep(cid:7)re5se).nAtalltipvaeirseimdureltcaonredoinugsswwheorlee-pceerlflocrumrreedntb-ectlawmepenreZcTo2r2dianngdsoZfTt2w3.oBip,sQiluaatnertaifliclLaNtivosntroefattheedcwoirtrhelpaitciroontooxfinm(e1m00br(cid:2)anMe)
theconnectivityofthenetwork.
activitybetweenpairsoflLNsbefore,during,andafterpicrotoxintreatment.Eachpairthatwastreatedwithpicrotoxinis
v
Tocharacterizethenatureofsynaptic shown(n(cid:7)5).C,RepresentativetraceofapairoflLNsthatswitchtheirmodeoffiringfromtonictoburstingasaresult
v
inputs to lLN s, we used a combination ofpicrotoxintreatment(n(cid:7)2).
v
of agonists and antagonists against neu-
rotransmitter receptors in both current- firm that lLN s in the adult circadian neural network possess
v
clampandvoltage-clampmode.Current-clampmodewasused nAChRsandthatthesereceptorsmediateexcitatorysynapticin-
tomonitormodulationofmembraneactivityinthelLNvsinthe putandsynchronyofrhythmicfiring.
contextofthefunctionalneuralnetwork,whereasvoltage-clamp GABA is a major neurotransmitter in the Drosophila CNS
modewasusedtodeterminethepresenceofunderlyingreceptors
(ffrench-Constantetal.,1991),mediatingfastinhibitorysynaptic
inthelLN sthemselves.WefoundthatlLN sreceiveexcitatory
v v transmission through the GABA receptor. This receptor has
cholinergicinputthroughnAChR(Figs.3,4D,E).Treatmentof A
beenshowntobeexpressedinLN sandhasbeenshowngeneti-
brainswithcholinergicreceptoragonists,acetylcholineandnic- v
callytoplayamajorroleintheregulationofarousalandsleepby
otine, enhances membrane activity, depolarizing the neurons
lLN sspecifically(Pariskyetal.,2008;Shangetal.,2008;Chunget
and increasing action potential firing rate, whereas treatment v
al.,2009).GABA-induceddecreasesinintracellularcalciumand
with ACh receptor antagonists, curare and (cid:1)-BuTX, inhibits Cl(cid:3) currents have been recorded in dissociated PDF neurons
membrane activity (Fig. 3). Voltage-clamp recordings in the
fromthelarvalandadultflybrain,respectively(Hamasakaetal.,
presenceofTTXrevealedthatACh-andnicotine-inducedcur-
2005;Chungetal.,2009),butpreviousstudieshavenotanalyzed
rentsoccurinlLN sthemselves,andthesecurrentsreversenear
v effectsofGABAonlLN membraneactivityinthecontextofthe
the equilibrium potential for nonselective monovalent cation v
intact circadian rest/arousal control network. We demonstrate
channels,asexpectedforcurrentsthroughnicotinicacetylcho-
thatGABAinhibitsthemembraneactivityoflLN s,whereasthe
linereceptors(Fig.4D,E).Ininsects,acetylcholineistheprimary v
ionotropic GABAR antagonist, picrotoxin, is excitatory (Fig.
excitatory neurotransmitter in the CNS (Sattelle et al., 1989;
5A,B,D,E).Incontrast,thestudybyHamasakaetal.showsno
GundelfingerandHess,1992),andnAChRsarewidelyexpressed
intheDrosophilabrain(Schusteretal.,1993).Thesereceptorsare rescueoftheinhibitoryeffectofGABAbypicrotoxinonPDF(cid:4)
knowntomediatefastsynaptictransmissioninKenyoncellsin LNvprecursors(Hamasakaetal.,2005)butdoesshowalleviation
the adult mushroom body (Su and O’Dowd, 2003; Gu and of GABA-induced inhibitory responses in these neurons by
O’Dowd,2006).Apreviousstudyhasalsoshownthatdissociated metabotropicGABABRantagonists.Althoughourstudiesdonot
PDF-positiveneuronsfromthelarvalDrosophilabrain,whichare excludearoleforGABA RsinlLN s,thediscrepancyoftheeffect
B v
developmentalprecursorsoftheLN s,expressnAChRsandex- of picrotoxin could be attributable to changes in different
v
hibitbothACh-inducedandnicotine-inducedincreasesinintra- GABARsubtypeexpressionatdifferentdevelopmentalstages.
cellularcalciumthataredependentonbothexternalsodiumand Throughanextensiveseriesofvoltage-clampexperimentswe
calciumconcentrations(Wegeneretal.,2004).Ourfindingscon- determinedthatGABAinducescurrentsinlLN sthatreverseat
v