Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.

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by  Augustin, Hrvoje

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Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster
Hrvoje Augustin1,MarcusJ. Allen2,Linda Partridge1
1InstituteofHealthyAgeing,andGEE,UniversityCollegeLondon-UCL
2SchoolofBiosciences,UniversityofKent
Correspondenceto:[email protected]
URL:http://www.jove.com/details.php?id=2412
DOI:10.3791/2412
Citation:Augustin H.,Allen M.J.,Partridge L.(2011).ElectrophysiologicalRecordingsfromtheGiantFiberPathwayofD.melanogaster.JoVE.47.
http://www.jove.com/details.php?id=2412,doi:10.3791/2412
Abstract
WhenstartledadultD.melanogasterreactbyjumpingintotheairandflyingaway.Inmanyinvertebratespecies,includingD.melanogaster,the
"escape"(or"startle")responseduringtheadultstageismediatedbythemulti-componentneuronalcircuitcalledtheGiantFiberSystem(GFS).
Thecomparativelargesizeoftheneurons,theirdistinctivemorphologyandsimpleconnectivitymaketheGFSanattractivemodelsystemfor
studyingneuronalcircuitry.TheGFSpathwayiscomposedoftwobilaterallysymmetricalGiantFiber(GF)interneuronswhoseaxonsdescend
fromthebrainalongthemidlineintothethoracicganglionviathecervicalconnective.Inthemesothoracicneuromere(T2)oftheventralganglia
theGFsformelectro-chemicalsynapseswith1)thelargemedialdendriteoftheipsilateralmotorneuron(TTMn)whichdrivesthe
tergotrochanteralmuscle(TTM),themainextensorforthemesothoracicfemur/leg,and2)thecontralateralperipherallysynapsinginterneuron
(PSI)whichinturnformschemical(cholinergic)synapseswiththemotorneurons(DLMns)ofthedorsallongitudinalmuscles(DLMs),thewing
depressors.Theneuronalpathway(s)tothedorsoventalmuscles(DVMs),thewingelevators,hasnotyetbeenworkedout(theDLMsandDVMs
areknownjointlyasindirectflightmuscles-theyarenotattacheddirectlytothewings,butrathermovethewingsindirectlybydistortingthe
nearbythoraciccuticle)(KingandWyman,1980;Allenetal.,2006).Thedi-synapticactivationoftheDLMs(viaPSI)causesasmallbutimportant
delayinthetimingofthecontractionofthesemusclesrelativetothemonosynapticactivationofTTM(~0.5ms)allowingtheTTMstofirstextend
thefemurandpropeltheflyofftheground.TheTTMssimultaneouslystretch-activatetheDLMswhichinturnmutuallystretch-activatetheDVMs
forthedurationoftheflight.TheGFpathwaycanbeactivatedeitherindirectlybyapplyingasensory(e.g."air-puff"or"lights-off")stimulus,or
directlybyasupra-thresholdelectricalstimulustothebrain(describedhere).Inbothcases,anactionpotentialreachestheTTMsandDLMs
solelyviatheGFs,PSIs,andTTM/DLMmotoneurons,althoughtheTTMnsandDLMnsdohaveother,asyetunidentified,sensoryinputs.
Measuring"latencyresponse"(thetimebetweenthestimulationandmuscledepolarization)andthe"followingtohighfrequencystimulation"(the
numberofsuccessfulresponsestoacertainnumberofhighfrequencystimuli)providesawaytoreproduciblyandquantitativelyassessthe
functionalstatusoftheGFScomponents,includingbothcentralsynapses(GF-TTMn,GF-PSI,PSI-DLMn)andthechemical(glutamatergic)
neuromuscularjunctions(TTMn-TTMandDLMn-DLM).Ithasbeenusedtoidentifygenesinvolvedincentralsynapseformationandtoassess
CNSfunction.
Protocol
1. Equipment and Materials
1. Theseexperimentsuseastandardelectrophysiologysetupcomprisedofastimulator,astimulusisolationunit,twomicroelectrodeamplifiers,
adataacquisitionsystemandacomputerwithcollectionsoftware.AdditionalequipmentincludesaFaradaycage,astereomicroscopeona
boomstand,avibrationisolationtable,alightsource,andarecordingplatform.
2. Fivemicromanipulatorsareused.Twomicromanipulatorsrequirefinecontrolsforpositioningtherecordingelectrodes,whiletheotherthree
micromanipulatorsonlyrequiregrosscontrolstopositionthetwostimulationelectrodesandthegroundelectrode.Themicromanipulatorfor
theDLMrecordingelectrodeisplacedatthetailendofthepreparation(leftofexperimenter)andthemicromanipulatorfortheTTMrecording
electrodeisplacedbetweentheexperimenterandthesideofthepreparation(slightlytotheleftoftheexperimenter).Thetwo
micromanipulatorsthatwillholdthesimulationelectrodesareplacedattheheadofthepreparation(rightofexperimenter).The
micromanipulatorforthegroundelectrodeisplacedatthefarsideofthepreparation
3. Pullglassrecordingmicroelectrodeswithresistancesof40-60MΩandstoreflatinadishsupportedbywax.Forstimulation,two
electrolytically(NaOH)sharpenedtungstenelectrodesareused.Atungstenwire,orathirdelectrolyticallyfabricatedelectrodeisusedasa
ground.Thestimulatingandgroundelectrodesarepreparedandattachedtothemicromanipulatorsbeforethestartoftheexperimental
sessionandneednotbereplacedforthedurationofthesession.
2. Preparing the D. melanogaster
1. Onceyourequipmentissetup,it'stimetopreparetheflies.AnesthetizethefliesbycoolingonthemiceorbyusingCO2.IfCO2isused,then
allowsufficienttime(about20mins)fortheeffectsofthegastowearoffpriortobeginningtheexperiment.
2. Useforcepstotransferthefliesgentlybytheirlegstoadishcontainingaplatformofsoftwaxslopedatanangleofapproximately45°.The
nextfourstepsaredoneunderadissectingmicroscopeawayfrom(butcloseto)therecordingequipment.
3. Thenextstepistosecuretheflyinwax.Orienttheflyventralsidedown,withitsanteriorfacingupwardsontheslope.Usingapairoffine
forceps,extendthelegsoutwards,inpairs,andpushthemintothewax.
4. Familiarizeyourselfwiththelocationofthemusclestoberecordedfrom:thedorsallongitudinalmuscle,orDLM,andthetergotrochanteral
muscle,orTTM.ThesubcuticularattachmentsitesoftheDLMscorrespondwiththeregionbetweenthethoracicmidlineandtheanterior
dorsalbristles(orsetae).TheTTMattachmentsitesarelocateddorsallyoftheposteriorandanteriorsupra-alarbristles.Makingsurethatthe
wingswillnotobstructaccesstotheDLMorTTMfibers,holdthewingsoutwardand'glue'themtothewax.
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5. Usingafinepairofforceps,pulltheproboscisoutwardcarefully,andsecureitbyimmersingitintothewax.Thisisacriticalstepthatrequires
somepracticesincetheproboscisissoftandiseasilydetachedfromtherestofthehead.Ifthathappens,discardtheflyandstartover.
Failuretosecuretheheadinthiswayleadstoproblemswheninsertingthestimulatingelectrodesthroughtheeyes.
3. Placing the Electrodes
1. Oncetheflyisanchoredtothewax,transferthedishwiththeattachedflyunderneaththestereomicroscopethatislocatedinsidetheFaraday
cage.Orienttheflysidewayswiththeheadoftheflytotherightoftheexperimenter.
2. Thenextstepistoinserttheelectrodes.Groundandstimulatingelectrodescanbeinsertedwithoutlookingthroughthemicroscope.Good
recordingsrelyonpreciseimpalement,soitisagoodideatopracticehandlingthemicromanipulators.Bringtheelectrodesclosetothesites
ofinsertionwiththehelpofmicromanipulatorstofacilitatetheirproperplacingandsubsequentrecordings.
3. Lowerthegroundelectrodeintotheposteriorendoftheabdomenusingtheadjustmentwheelsonthemicromanipulator.Toplacethe
sharpenedtungstenstimulatingelectrodesinthebrain,usethemicromanipulatortopositionthetipofoneoftheelectrodessoitjusttouches
oneoffly'seyes.Dothesamewiththeothersobothelectrodesarejusttouchingtheoutsideofeacheye.Thenpushtheelectrodes,inturn,
througheacheyesothetipsoftheelectrodesreachthebrainsituatedatthebackoftheheadcapsule(about2-3mm).
4. CorrectlyplacedelectrodeswillactivatetheGiantFiberSystem.Totestthatthestimulatingelectrodesareplacedcorrectly,applyashort
(0.03ms)stimulusof30-60Vacrossthestimulatingelectrodes,andlookformovementofthewingsandtwitchesoftheflight/legmuscle'
5. Thenextstepistoback-filltheglassmicroelectrodeswith3MKClusingaHamiltonorheat-pulledplasticsyringe,andplacethemintothe
fine-controlmicromanipulators.Properlyinsertedmicroelectrodescanbeusedforseveralroundsofexperiments.
6. ThefirstrecordingelectrodewillbeinsertedintoaDLMfiber.TherearetwobilaterallysymmetricDLMs;eachoneiscomposedofsix
individualmusclefibers.Therecordingscanbedonefromanyofthesixfibers;however,themostcommonlyusedareDLMfibers45aand
45bduetotheirgoodaccessibilitythroughthedorsalsideofthethoraciccuticle,andthefactthatbothfibersareinnervatedbythesame
motorneuron.
7. Usingthemicromanipulatoronthesidefarthestfromyou,insertarecordingelectrodeintoDLMfiber45aorb.Theslopeoftheplatform
allowstheDLMelectrodetoenterthedorsalcuticleata~60-90°angle,whichaidspenetration.Usethesoftwareinoscilloscopemodeand
lookatthecomputermonitorwhileinsertingrecordingelectrodesintothethorax.Whentheelectrodehasenteredamusclethebaselinewill
droptonearzerooranegativevalue.Testwithasinglestimulustoseeifyoucanobservethemuscleresponse.
8. InserttheotherrecordingelectrodeintotheTTMclosesttoyou.Thiselectrodeisinsertedlaterally,infrontofyou,duetothelocationofthe
muscleattachmentsite.Againobservethemonitorwhiledoingthisandtestwithasinglestimulusoncethetraceindicatestheelectrodeisin
themuscle.
4. Stimulation and Recording
1. Youarenowreadytobeginstimulatingthebrainandrecordingresponsesfromthelegandflightmuscles.Applyashort(0.03ms)stimulus
acrossthestimulatingelectrodesstartingat30Vandincreasingto60Vuntilyouobservearesponse(i.e.amuscletwitch,andamusclecell
depolarizationasobservedonthecomputermonitor).Fortheremainderoftheexperiment,setthevoltage5-10Vabovetheresponse
threshold.
2. Tomeasureresponselatency,giveatleast5singlestimuliwitha5secondrestperiodbetweeneachstimulus.
3. Determinethe"frequencyoffollowing"byprovidingtrainsofstimuliatdifferentrates.Typically10trainsof10stimuliaregivenat100Hz
(10msbetweeneachstimulus),200Hz(5msbetweeneachstimulus)and300Hz(3msbetweeneachstimulus).Allowarestperiodof2
secondsbetweeneachtrainofstimuli.
5. Results: Response Latencies and Frequency of Following in the Giant Fiber Pathway
1. Theresponselatencyisthetimebetweenstimulationofthebrainanddepolarizationofthemuscle.Thisfigurecomparestheresponse
latenciesforDLMandTTMtoasinglestimulus.Latenciesbetween0.7and1.2msfortheGF-TTMpathwayandbetween1.3and1.7msfor
theGF-DLMpathwayindicateahealthypreparationandproperrecordingtechnique.Thelatenciescanvarywithgenotype,genetic
background,temperatureandage.
Figure1(AandB).RepresentativetracesshowingresponsesrecordedfromtheTTMsandDLMsfollowingasinglestimulusappliedtothe
brain.
2. Asshownhere,recordingsfromtheTTMshowmorevariabilityintermsofamplitudeandshapeofthepostsynapticpotential(PSP)compared
tothosefromthelargeDLMfibers;thisincreasedvariabilityisduetothesmallsizeoftheTTMmusclefibers.Thisvariability,however,does
notaffecttheresponselatencyvaluesfortheGiantFiber-to-TTMpathway.
Figure1(CandD).Further'responselatency'tracesfrom4individualfliesforboththeTTMandDLM.NoteTTMtracesexhibitvariabilityin
PSPshapebutresponselatencyisunaffected.ForDLMthereislessvariabilityinPSPshape.
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3. Comparethe"frequencyoffollowing"at100Hz,200Hz,and300Hzbycalculatingtheproportionofsuccessfulresponses(outof10)forboth
DLMandTTMpathwaysateachstimulationfrequency.At100Hz,bothTTMandDLMfollowthestimuli1:1.Atstimulationfrequenciesabove
100Hz,theDLMresponsesstarttoshowfailuresbecausetheintermediarychemicalsynapsebetweentwointerneuronsdoesnothave
sufficienttimetorecoverbetweenstimuli.TheTTMresponses,however,remain1:1withstimulievenbeyond300Hz.
Figure2.Representativetracesshowingthe"frequencyoffollowing"recordings.At100Hz,bothTTMsandDLMsrespondtoall10stimuli
(left).At200Hz,theDLMresponsesstarttofail(asterisk).
6. Representative Results
Wildtypeshort-latencyresponses(stimulatedelectrodesareplacedintheeyes,bypassingsensoryreceptorsandtriggeringtheGFcircuit
directly)dependongenotype,geneticbackground,temperatureandage,andrangebetween0.7and1.2msfortheGF-TTMpathwayand1.3
and1.7msfortheGF-DLMpathway(TanouyeandWyman,1980;ThomasandWyman,1984;EngelandWu,1992;AllenandMurphey,2007;
Phelanetal.,2008;Augustinetal.,unpublished).ThisveryshortTTMlatencyisduetotherobustGF-TTMnelectrochemicalsynapseofthe
monosynapticpathwayandthelongerDLMlatencyoccursbecauseofthedisynapticnatureofthepathwayaswellasthepresenceofachemical
synapse(PSI-DLMn).Intermediate-andlong-latencyresponses(>3ms)resultfromtheactivationoftheGFafferentsandareachievedeitherby
usingalowerintensitystimulationorprovidingavisual("light-off")signal.At100HzbothTTMandDLMshouldfollowthestimuli1:1.Above100Hz
DLMresponseswillstarttoshowfailuresasthechemicalsynapsebetweenPSIandtheDLMnsdoesnothavesufficienttimetorecoverbetween
stimulilessthan10msapart.TTMresponses,however,willremain1:1withstimulievenbeyond300Hz(TanouyeandWyman,1980;Engeland
Wu,1992;Allenetal.,2007;Martinezetal.,2007).MutationsintheshakBgene,encodingaDrosophilagapjunctionchannel("innexin"),
significantlyincreasetheresponselatencyoftheGF-TTMpathway(~1.5ms)whiletheGF-DLMbranchisunresponsive(AllenandMurphey,
2007;Phelanetal.,2008).Themutantresponsecanberestoredbystimulatingthoracicgangliadirectly,demonstratingthatthedelayedeffectis
notduetodisruptedneuromusculartransmission.Theabilitytofollowhighfrequencystimulationisalsoimpairedinthesemutantscomparedto
wildtypeflieswheretheGF-DLMandGF-TTMpathwaysareusuallyabletofollow10stimuliwith1:1ratioupto100Hzand300Hz,respectively.
Itisimportanttonotethatthesefrequenciesareconsiderablyabovenormalstimulationfrequenciesreceivedbythecontractingmusclesduring
thesustainedflight(3-10Hz)(HummonandCostello,1989).
AnotherparameterusedtodescribethestabilityoftheGFSoutputsisthe"refractoryperiod",ortheminimaltimebetweentwinstimuluspulses
thatstillproducestworesponsesfromthemuscle.Therefractorytimevariesbetween1-4msforTTMsand7-15msforDLMs.Thecomparatively
longrefractoryperiodforDLMsisduetorelativelylabilechemicalsynapsesatthePSI-DLMnjunction(TanouyeandWyman,1980;Gorczycaand
Hall,1984;EngelandWu,1992;Banerjeeetal.,2004;AllenandGodenschwege,2010).
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Discussion
Oneofthemostimportantthingsonehastopayattentiontowhentryingtoobtainhighqualityrecordingsistheproperorientationandhealthof
thepreparation.Ideally,theflyshouldstillbealiveattheendoftherecordingsessionandresponsivetoelectricalstimuli.Fortherecording
electrodestomostefficientlypenetratethethoracicexoskeleton,theflyshouldbegluedtothesurfaceinsuchawayastoformarightanglewith
theelectrodes;ifnecessary,theinsertionofelectrodescanbefacilitatedbyremovingaportionofthedorsalthoraciccuticlewithatungsten
scalpelthusexposingtheDLMflightmuscle(thisstepoffersanadditionaladvantageofmakingitharderforthetipsofglasselectrodestobreak).
Also,thecaremustbetakentoavoidpushingtheelectrodesthroughthesubcuticularlylocatedDLMsandTTMs.Theheadoftheflyshouldbe
wellsecuredtoallowforthestimulatingelectrodestobeproperlyinsertedintothebrainandtopreventthemfrombeingpulledoutduringthe
recordingsession.
Duetoitssizeandwell-describedmorphology,theGFSrepresentsoneofthemostaccessibleneuronalpathwaysinDrosophila.The
permeabilityofelectricalsynapsestosmallmolecularweighttracerdyesallowsforthevisualisationofelectricallycoupledneurons,andseveral
availableGAL4linesmakeitpossibletomanipulategeneexpressionlevelsinasubsetofcellsorcellgroups(Jacobsetal.,2000;Allenetal.,
2006)Inadditiontotheabovementionedadvantages,bothafferentandthoraciccomponentsofthecircuitdisplaypropertiessuchashabituation,
spontaneousrecoveryanddishabituation,makingtheDrosophilaGFSaconvenientmodelsystemforstudyingneuronalplasticity(EngelandWu,
1996).
Disclosures
Noconflictsofinterestdeclared.
Acknowledgements
ThisworkwassupportedbyaWellcomeTrustgranttoL.P.
References
1. Allen,M.J.,Godenschwege,T.ElectrophysiologicalRecordingsfromtheDrosophilaGiantFiberSystem.In:BingZhang,MarcR.Freeman,
ScottWadde,DrosophilaNeurobiology:ALaboratoryManual(1stLabed.),ColdSpringHarborPress,pp.(2010).
2. Allen,M.J.,Godenschwege,T.A.,etal.Makinganescape:developmentandfunctionoftheDrosophilagiantfibresystem.SeminCellDev
Biol17(1):31-41(2006).
3. Allen,M.J.,andMurphey,R.K.ThechemicalcomponentofthemixedGF-TTMnsynapseinDrosophilamelanogasterusesacetylcholineas
itsneurotransmitter."EurJNeurosci26(2):439-445(2007).
4. Banerjee,S.,Lee,J.etal.Lossofflightandassociatedneuronalrhythmicityininositol1,4,5-trisphosphatereceptormutantsofDrosophila.J
Neurosci24(36):7869-7878(2004).
5. Engel,J.E.,andWu,C.F.Interactionsofmembraneexcitabilitymutationsaffectingpotassiumandsodiumcurrentsintheflightandgiant
fiberescapesystemsofDrosophila.JCompPhysiolA171(1):93-104(1992).
6. Engel,J.E.,andWu,C.F.AlteredhabituationofanidentifiedescapecircuitinDrosophilamemorymutants.JNeurosci16(10):3486-3499.
7. Gorczyca,M.andHall,J.C.IdentificationofacholinergicsynapseinthegiantfiberpathwayofDrosophilausingconditionalmutationsof
acetylcholinesynthesis.JNeurogenet1(4):289-313(1984).
8. Hummon,M.R.,andCostello,W.J.GiantfiberactivationofflightmusclesinDrosophila:asynchronyinlatencyofwingdepressorfibers.J
Neurobiol20(6):593-602(1989).
9. Jacobs,K.,Todman,M.G.,etal.Synaptogenesisinthegiant-fibresystemofDrosophila:interactionofthegiantfibreanditsmajor
motorneuronaltarget.Development127(23):5203-5212(2000).
10. King,D.G.,andWyman,R.J.AnatomyofthegiantfibrepathwayinDrosophila.I.Threethoraciccomponentsofthepathway.JNeurocytol
9(6):753-770(1980).
11. Martinez,V.G.,Javadi,C.S.,etal.Age-relatedchangesinclimbingbehaviorandneuralcircuitphysiologyinDrosophila.DevNeurobiol
67(6):778-791(2007).
12. Miller,A.TheinternalanatomyandhistologyoftheimagoofDrosophilamelanogaster.In:M.Demerec,Editor,BiologyofDrosophila(2nded.),
(Hafner,NewYork)pp.502-503(1965).
13. Phelan,P.,Goulding,L.A.,etal.MolecularmechanismofrectificationatidentifiedelectricalsynapsesintheDrosophilagiantfibersystem.
CurrBiol18(24):1955-1960(2008).
14. Power,M.E.Thethoracico-abdominalnervoussystemofanadultinsect,Drosophilamelanogaster.JCompNeurol88(3):347-409(1948).
15. Tanouye,M.A.andWyman,R.J.MotoroutputsofgiantnervefiberinDrosophila."JNeurophysiol44(2):405-421(1980).
16. Thomas,J.B.andWyman,R.J.MutationsalteringsynapticconnectivitybetweenidentifiedneuronsinDrosophila.JNeurosci4(2):530-538
(1984).
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