University of Pennsylvania ScholarlyCommons

Departmental Papers (ASC) Annenberg School for Communication

2006

The Dynamics of Recycled Acetylcholine Receptors at the Neuromuscular Junction in vivo

Emile Bruneau University of Pennsylvania, [email protected]

Mohammed Akaaboune

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Recommended Citation Bruneau, E., & Akaaboune, M. (2006). The Dynamics of Recycled Acetylcholine Receptors at the Neuromuscular Junction in vivo. Development, 133 4485-4493. https://doi.org/10.1242/dev.02619

This paper is posted at ScholarlyCommons. https://repository.upenn.edu/asc_papers/560 For more information, please contact [email protected]. The Dynamics of Recycled Acetylcholine Receptors at the Neuromuscular Junction in vivo

Abstract At the peripheral neuromuscular junction (NMJ), a significant number of nicotinic acetylcholine eceptr ors (AChRs) recycle back into the postsynaptic membrane after internalization to intermingle with not-yet- internalized `pre-existing' AChRs. However, the way in which these receptor pools are maintained and regulated at the NMJ in living animals remains unknown. Here, we demonstrate that recycled receptors in functional synapses are removed approximately four times faster than pre-existing receptors, and that most removed recycled receptors are replaced by new recycled ones. In denervated NMJs, the recycling of AChRs is significantly depressed and their removal rate increased, whereas direct muscle stimulation prevents their loss. Furthermore, we show that protein tyrosine phosphatase inhibitors cause the selective accumulation of recycled AChRs in the peri-synaptic membrane without affecting the pre-existing AChR pool. The inhibition of serine/threonine phosphatases, however, has no effect on AChR recycling. These data show that recycled receptors are remarkably dynamic, and suggest a potential role for tyrosine dephosphorylation in the insertion and maintenance of recycled AChRs at the postsynaptic membrane. These findings may provide insights into long-term recycling processes at less accessible synapses in the central nervous system in vivo.

Keywords recycling, receptor dynamics, half-life, phosphorylation, synaptic activity

Disciplines Biological Psychology | Communication | Molecular and Cellular Neuroscience | Neurology | Neurosciences | Social and Behavioral Sciences

This technical report is available at ScholarlyCommons: https://repository.upenn.edu/asc_papers/560 2000; Hayashietal.,LeeMalinow andMalenka, potentiation orlong-termdepression(Carrolletal.,1999;Ehlers, role inregulating synapticchangesthatleadtoeitherlong-term been extensively studied,andhasbeenfoundtoplayanimportant and maintenanceofionotropicreceptorsincentralsynapses has determined. AChR poolsaremaintainedandregulated remainstobe existing’ AChRs) (Bruneauetal.,2005).However, theway these retain theirinitialfluorescenttag(referredtohereafteras‘pre- intermingle withAChRs thathave notyetbeeninternalizedand instead returnbackintothepostsynapticmembranewherethey a significantnumberofinternalizedAChRs arenotdegraded, but time. Recentwork fromourlaboratoryhasshown, however, that maintaining AChR densityinthepostsynapticmembraneover insertion ofnewly synthesizedreceptorswould beresponsiblefor lysosomes (GardnerandFambrough, 1979).Inthis scheme, membrane untilthey areinternalizedanddegraded inthe acetylcholine receptors(AChRs) remainstableinthepostsynaptic neuromuscular junction(NMJ),itwas longassumedthat Luscher etal.,1999;Park etal.,2004).Atthe peripheral (Contractor andHeinemann,2002;Ehlers,2000;Leeetal.,2004; endosomal compartmentstothepostsynapticmembrane isoxazole propionicacid(AMPA) receptorsfromintracellular recruitment ofrecycled potentiation, synapticstrengthisenhancedbyanincreased crucial roleinsynapticplasticity. For example, duringlong-term At centralsynapses,recycling ofpostsynapticreceptors playsa INTRODUCTION KEY WORDS:Recycling,Receptordynamics,Half-life,Phosphorylation,Synapticactivity term recyclingprocessesatlessaccessiblesynapsesinthecentralnervoussystemvivo. the insertionandmaintenanceofrecycledAChRsatpostsynapticmembrane.Thesefindingsmayprovideinsightsintolong- These datashowthatrecycledreceptorsareremarkablydynamic,andsuggestapotentialrolefortyrosinedephosphorylationin affecting thepre-existingAChRpool.Theinhibitionofserine/threoninephosphatases,however, hasnoeffect onAChRrecycling protein tyrosinephosphataseinhibitorscausetheselectiveaccumulationofrecycledAChRsinperi-synapticmembranewithou depressed andtheirremovalrateincreased,whereasdirectmusclestimulationpreventsloss.Furthermore,weshowthat most removedrecycledreceptorsarereplacedbynewones.IndenervatedNMJs,therecyclingofAChRsissignificantly that recycledreceptorsinfunctionalsynapsesareremovedapproximatelyfourtimesfasterthanpre-existingreceptors,andtha which thesereceptorpoolsaremaintainedandregulatedattheNMJinlivinganimalsremainsunknown.Here,wedemonstrate the postsynapticmembraneafterinternalizationtointerminglewithnot-yet-internalized‘pre-existing’AChRs.However, in theway At theperipheralneuromuscularjunction(NMJ),asignificantnumberofnicotinicacetylcholinereceptors(AChRs)recyclebacki Emile G.BruneauandMohammedAkaaboune* invivo neuromuscular junction The dynamicsofrecycledacetylcholinereceptorsatthe Development 133,4485-4493(2006)doi:10.1242/dev.02619 Accepted 7September 2006 *Author forcorrespondence (e-mail:[email protected]) 48109, USA. Program, UniversityofMichigan,830NorthAvenue, AnnArbor, MI Department ofMolecular, CellularandDevelopmentalBiologyNeuroscience The involvement ofphosphorylationinthetrafficking, insertion -amino-5-hydroxy-3-methyl-4- water immersionobjectives (20 epifluorescence microscope,andNMJs wereviewed underacoverslip with anesthetized mousewas placedonitsbackthestageofacustomized Lichtman etal.,1987;van MierandLichtman,1994).Briefly, the imaging wereperformedaspreviously described(Akaabouneetal.,1999; respectively, perkgbodyweight).Sternomastoidmuscleexposure andNMJ intraperitoneal injectionofketamine andxylazine(KX;87mg13mg, NSA, HarlanSpragueDawley, Indianapolis,IN)wereanesthetizedwithan Committee ontheUseandCareofAnimals.Adultfemalemice(20-27 g; All animalusagefollowed methodsapproved bytheUniversity ofMichigan Live animalimaging MATERIALS ANDMETHODS synaptic accumulationofrecycled AChRs. serine/threonine phosphataseinhibitors,causetheaberrantperi- addition, wefoundthattyrosinephosphataseinhibitors,but not the insertionofrecycled AChRs and increasestheirremoval rate.In of functionalsynapses,anddemonstratethatdenervation decreases than pre-existing receptorsfromthesamepostsynaptic membrane Here, wereportthatrecycled receptorsareremoved morequickly receptor poolsinthepostsynapticmembrane(Bruneauetal.,2005). previously usedtoselectively identifyrecycled andpre-existing pre-existing AChRs usingasequential labelingmethodthatwehad dephosphorylation inAChR recycling, wemonitoredrecycled and AChR pools,andthepotentialroleofphosphorylation/ investigated. AChR recycling attheNMJinvivo hasnotpreviously been however, theroleofphosphorylation/dephosphorylationevents in 2004; MeiandSi,1995;Wallace, 1994;Wallace etal.,1991); vivo andinvitro(Gradyetal.,2003;Huganir1984;Li phosphatases areinvolved inAChR anchoringandclusteringin 2002). AttheNMJ,alarge bodyofwork demonstratesthat imaging session. sutured aftereachsessionandallowed tofullyrecover beforethenext of theimagingsessions.For imagingatmultipletimepoints,themousewas Burnaby, BC, Canada).Micewereintubatedandventilated fortheduration Optical AnalysisCorporation,NH) andadigitalCCDcamera(RetigaEXi, In ordertoinvestigate thedynamics ofrecycled andpre-existing and 60 RESEARCH ARTICLE UAPO 0.7NA, OlympusBW51, 4485 t nto . t

DEVELOPMENT muscle was incubatedwithunlabeledBTX (5 al. (Bruneauetal.,2005). biotin-streptavidin dissociationwereestablishedpreviously byBruneauet al., 2005;LingleandSteinbach,1988).Allcontrolsforthespecificityof and thesearesufficient toallow normalsynaptictransmission(Bruneauet significant numberofreceptorsintheNMJarenew, functionalreceptors, pools. Itisworth notingthatatthetime ofrecycled AChR labeling,a receptors (retainingtheirinitialstreptavidin) fromtherecycled receptor this way, wewereabletodistinguishthepre-existing BTX-biotin-labeled subsequent timepoints,thesamesynapseswererelocatedandimaged.In synapses werethenimaged(IPLABsoftware, Scanalytics,VA). At erroneous estimationofreceptorremoval. Thedoublylabeledsuperficial after theextensive washing. Thislabelingprocedurewould prevent any AChRs ofany residualfluorescentstreptavidin thatremainedinthemilieu to thesternomastoidmuscleprevent the(unlikely) binding torecycled solution. Abriefchaseofunlabeledstreptavidin (10minutes)was added muscle was washed outcontinuouslyfor15minuteswithRinger’s recycled backtothemusclesurface asAChR-BTX-biotin complexes. The muscle re-exposed andbathedwithstrept-594tolabeltheAChRs thathad to 4dayslater, themousewas re-anesthetized,andthe sternomastoid indicated thatallbiotinsiteswereinitiallysaturatedwithstrept-488.Three muscle, andthesynapsesimaged.Theabsenceofstrept-594staining (strept-594; red;10 saturated, asecond,differently-colored label,streptavidin-Alexa 594 Probes) tosaturateallbiotinsites.To ensurethatallbiotinsiteswere conjugated toAlexa 488(strept-488;green;10 Probes, Eugene,OR)tolabelAChRs, andthenwithstreptavidin substituted bungarotoxin (BTX)-biotin(5 al., 2005).Briefly, thesternomastoidmusclewas bathedfirstwithfully The labelingprotocolwas performedaspreviously described(Bruneauet Labeling ofdistinctAChRpools 4486 muscle fibers.Mice werecontinuouslyventilated andmaintainedunder seconds) elicitedmaximaltwitching andthereforeactionpotentialsinall msecond bipolarpulsesof6-9Vat 10Hzfor1seconddurationevery 2 platinum wiresplacedeitherside of themuscle.Thestimuluspulses(3 was directly stimulatedwithaGrassSD5stimulatorconnectedtotwo immediately afterrecycled receptorlabelingindenervated NMJs,themuscle time. the decreaseinfluorescenceofbothAChR poolswas monitoredover synapses wereimaged,theirfluorescenceintensitiesmeasured, and (to labelallreceptorsthathadrecycled over thattime);then,superficial exposed andsynapseslabeledwithasinglesaturatingdose ofstrept-594 ofstrept-488.Threedayslater, thesternomastoidmusclewas dose earlier was labeledwithBTX-biotin,followed byasinglesaturating denervated muscle,thesternomastoidmuscleofmicedenervated 6-8days to innervated synapses. intensity assayed.For comparison,asimilarlabelingprotocol was applied (to labelrecycled AChRs), synapseswereimagedandthefluorescence measured. Thesternomastoidmusclewas bathedagainwithfreshstrept-594 anaesthetized, thesamesynapseswereimagedandlossoffluorescence were imagedasdescribedabove. Threedayslater, theanimalwas re- then withasinglesaturatingdoseofstrept-594,andsuperficialsynapses sternomastoid musclewas exposed andlabelledfirstwithBTX-biotin, membrane, micedenervated 6-8daysearlierwereanesthetizedandthe denervation onthenumber ofreceptorsrecycled atthepostsynaptic sternomastoid nerve toprevent re-innervation. To determinetheeffect of Sternomastoid musclesweredenervated byexcising a5mmpieceofthe Surgical procedures fluorescence losswas monitoredover several days. saturating doseofstrept-488(green).Superficialsynapseswereimagedand 20 minutes),sothatsynaptictransmissionremainedfunctional,followed bya receptors werelabeledwithBTX-biotinatasub-saturatingdose(5 AChRs weresaturated). Four to5daysafterinitiallabeling,newly inserted surface receptors(aseconddoseoffluorescent BTXwas usedtoverify thatall To determinethelossrateofnewly synthesizedreceptors,the sternomastoid To determinetheeffect of muscleactionpotentialsonAChR turnover, To determinetheremoval rateofrecycled andpre-existing AChRs in RESEARCH ARTICLE g/ml, 10-20minutes)was addedtothesternomastoid g/ml, 1.5hours)tosaturateall g/ml, 1hour;Molecular g/ml, 3hours;Molecular g/ml for adjusted withPhotoshoptomaximallyresolve intracellular fluorescent spots. fluoview) andimaged.The Muscle sectionswerescannedwithaconfocalmicroscope(Olympus an anti-ratsecondaryantibodyconjugatedtoAlexa 647(Molecular Probes). Developmental StudiesHybridomaBank,Iowa University, IA),followed by thenbathedwithmonoclonalanti-receptor antibody(mAb35; and phosphatase activity (RediPlate to adirectfluorescence-basedassayfordetectingserine/threonine homogenized inRIPA buffer (Sigma).Thesupernatantwas thensubjected had beenbathedwithokadaicacidwas removed fromthemouseand acid invivo, attheendofimagingsessionsternomastoidmusclethat inhibit serine/threoninephosphatases.To verify theeffectiveness ofokadaic with excitation at355±20nmandemission460±12.5nm. emitted byDiFMUwas measuredusing afluorescencemicroplatereader, treated anduntreatedmusclewereaddedtothewells.Thefluorescence generated. DiFMUPwas thenfullysolubilized andhomogenatesfrom difluoro-4-methyl-umbelliferyl phosphate),fromwhichDiFMUis and withthefluorogenicserine/threoninephosphatesubstrateDiFMUP(6,8- with inhibitorsofphosphatasesotherthanserine/threoninephosphatases, phosphatases PP1andPP2Awereaddedtoa96-wellmicroplatepreloaded instructions. Briefly, appropriatebuffers fortheserine/threonine phosphatases AssayKit;MolecularProbes),accordingtothemanufacturer’s For allagentstested,500 Pharmacology number ofAChRs. relatively trivial togetanaccuratequantitative measurementoftherelative saturation andthattheimagepixel intensitywas notsaturated,itwas and strept-488).Thus,aslongweverified thatlabelinghadreached that itinvolves repetitive imagingofthesamefluorescentligands(strept-594 key featureofthequantitative imagingapproachusedinthecurrentstudyis sessions, bycalibratingtheimageswithanon-fading referencestandard.A and temporalchangesinthelightsourcecamerabetweenimaging incorporates compensationforimagevariation thatmaybecausedbyspatial colleagues (Turney etal.,1996),withminormodifications.Thistechnique a quantitative fluorescenceimagingtechnique,asdescribedbyTurney and The fluorescenceintensityoflabeledreceptorsatNMJswas assayedusing Quantitative fluorescence imaging exposed muscle. of theexperiment. To minimizedehydration,acoverslip was placedover the anesthesia byintraperitonealinjectionsofKXevery 2hoursfortheduration this, thesternomastoid muscleoflive micewas labeled withBTX- (receptors retainingtheirstrept-488 afterinitiallabeling).To test receptors areasstableinthe synapse aspre-existing receptors (Bruneau etal.,2005),andwe wanted toknow whetherrecycled number ofreceptorsrecycle backtosynapsesafterinternalization Recent work fromourlaboratory hasshown thatasignificant NMJ oflivemice Recycled andpre-existing AChRremoval atthe RESULTS sections (20 the sternomastoidmuscleremoved andlongitudinally sectioned.Muscle animal was perfusedtranscardiallywith2%paraformaldehyde(PFA) and sternomastoid muscletolabelrecycled AChRs. Two to3dayslater, the later, theanimal was re-anaesthetizedandstrept-594was addedtothe single saturatingdoseofstrept-488(10 The sternomastoidmusclewas saturated withBTX-biotinfollowed bya Confocal microscopy (Madhavan etal.,2005).Okadaicacid(10-100 of 1:50for10minutesbeforeaddingittothesternomastoidmuscle prepared bymixing1.7%H St Louis,MO)andpervanadate (5-10mM).Pervanadate solutionwas phosphatase activity, weusedtwo agents:phenylarsine oxide(2mM;Sigma, intubated withaventilator toavoid asphyxiation.To inhibittyrosine sternomastoid musclefor9hours.Duringtheexperiment, theanimalwas m) wereblocked with10%BSAand0.5%Triton X-100, l ofastocksolutionwas placeddirectlyontothe 2 O z -stacks werethencollapsedandthecontrast 2 and sodiumorthovanadate (Sigma)inaratio TM 96 EnzChekserine/threonine g/ml, 3hours).Threeto4days Development 133(22) M; Sigma)was usedto

DEVELOPMENT internalized (Bruneauetal.,2005).Pre-existing AChRs thatretain rather onlyafterthecomplex (AChR-BTX-biotin/strept-488) is BTX-biotin doesnotoccurspontaneouslyonthemusclesurface, but immediately (day0).Thedissociationofstreptavidin fromAChR- strept-488 labelbut retainedBTX-biotin,andimagesweretaken strept-594 was addedtolabelrecycled receptorsthat hadlosttheir then allowed torecover. Threeto4daysaftertheinitiallabeling, that allbiotinsitesweresaturatedwithstrept-488.Theanimalwas superficial synapsesshowed nostainingwithstrept-594, indicating was exposed tothedifferently-colored strept-594.Imagesof all biotinsites.To ensurethatallbiotinsiteswerelabeled,themuscle biotin, followed byasinglesaturatingdoseofstrept-488 tosaturate Recycled AChRdynamicsinvivo Scale bars,20 receptors are removed significantlyfasterthanpre-existing receptors. the meanpercentage offluorescence intensity±s.d.Notethatrecycled all junctionsbytheapproach showninB.Eachdatapointrepresents the densityofAChRs.( initial imaging.Pseudo-colorimages provide alinearrepresentation of fluorescence intensityofeach AChRpoolwasnormalizedto100%at labeling ofrecycled AChRs (d0),and2dayslater(d2).Thetotal assayed forfluorescence intensitiesimmediatelyafterstrept-594 existing andrecycled AChRslabeledwithdifferent fluorophores were overlay) AChRsare intermingledinthepostsynapticmembrane.( (retaining theirstrept-488 afterinitiallabeling;green andyellow branch showingthatrecycled (red andyellowoverlay) andpre-existing selectively labelrecycled AChRs.( first withBTX-biotin/strept-488, then3-4dayslaterwithstrept-594 to rates from musclewaslabeled thesamesynapse.Thesternomastoid Fig. 1.Recycledandpre-existing AChRsare removed atdifferent m. C ) Graphsummarizingtheresults obtainedfrom A ) Highresolution imageofaNMJ B ) Pre- to know whetherrecycled andpre-existing receptors, aftertheir membrane but turnover atsuchdifferent rates(Fig.1), we wanted and pre-existing receptors intermingleatthesamepostsynaptic (Akaaboune etal.,1999;Bruneau etal.,2005).Becauserecycled synapses areinternalizedin intracellularendocytic vesicles We have previously shown thatreceptorsareremoved from intracellular vesiclesafter internalization Targeting ofrecycled AChRstostablefluorescent postsynaptic membrane. receptors matchesthelossofrecycled receptorsfromthe (Fig. 2A,B).Thisindicatesthattheinsertionofnew recycled to 99%(±17s.d., re-labeled withnew strept-594,thefluorescenceintensityreturned 36% (±6s.d., days. At2and3days,theremainingfluorescenceintensitieswere their originalfluorescence,whichcorrespondstoahalf-lifeof~1.3 594) was 35%(±6s.d.,n fluorescence remainingfromtherecycled pool(labeledwithstrept- measured thefluorescenceintensityat2days,wefound the that hadbeenrecycled over thegiven timeperiod.Whenwe bathed withasaturatingdoseoffreshstrept-594tolabelreceptors measured ateachdatapoint.Thesternomastoidmusclewas then synapses werere-imagedandthefluorescenceofeachsynapse the animalwas allowed torecover. Two dayslater, thesame as describedabove. Thesuperficialsynapseswerethenimaged,and with asaturatingdoseofstrept-594tolabeltherecycled receptors, the animalwas anaesthetizedandthesternomastoidmusclebathed followed byasaturatingdoseofstrept-488.Threeto4dayslater sternomastoid muscleofthreemicewas labeledwithBTX-biotin insertion ofnew recycled receptorsover thesametimeperiod.The membrane, wenext examined whetherthislossismatchedbythe receptors (P synthesized receptorsissignificantlyslower thanforrecycled after 2days.Thesedataindicatethattheremoval rateofnewly of theoriginalfluorescenceafter1day, andto69%(±10s.d.,n from newly synthesizedreceptorsdecreasedto79%(±5s.d.,n then re-imaged1and2dayslater. We foundthatthefluorescence synthesized receptors.Thesuperficialsynapseswereimagedand was bathedwithalow doseofBTX-biotin/strept-488 tolabelnewly allowed torecover. Four to5dayslater, thesternomastoidmuscle sternomastoid musclewas saturatedwithBTXandtheanimal receptors andstrept-488tolabelrecycled AChRs. obtained ifstrept-594was usedinitiallytolabelpre-existing 54% (±4s.d., 3 daysthefluorescencehaddecreasedto68%(±9s.d.,n original fluorescenceafter1day(half-life~4days),and2 biotin/strept-488 decreasedtoonly84%(±6s.d., same times,thefluorescenceintensityofAChRs labeledwithBTX- fluorescence, respectively. However, atthesamesynapsesover the with BTX-biotin/strept-594)decreasedto59%(±5s.d., the fluorescenceintensityofrecycled receptors(receptorstagged then theirfluorescenceintensitiesassayed.We foundthatafter1day, pre-existing AChRs werere-imaged,onceormultipletimes,and after labelingrecycled AChRs withstrept-594,bothrecycled and intermingled inthesamepostsynapticmembrane.Oneto3days time. Asshown inFig.1A,recycled andpre-existing AChRs and thechangesintheirfluorescenceintensitiesmonitoredover labeled withBTX-biotin/strept-594couldthenbeimagedseparately, BTX-biotin/strept-488 aftertheinitiallabelingandrecycled AChRs Given therapidlossofrecycled receptors fromthepostsynaptic To examine the removal rateofnewly inserted receptors,the <0.0001). n =15), respectively (Fig.1B,C).Similarresultswere n =20) and16%(±2s.d., n=10) oftheinitialrecycled AChR fluorescence =17) oftheoriginalfluorescence.When RESEARCH ARTICLE n =15) oftheoriginal n =20) ofthe =10) and n =25) of 4487 =14) =7)

DEVELOPMENT postsynaptic membrane oftheNMJ. days were replaced by newlyrecycled receptors re-inserted intothe Note thatnearlyalloftheAChRs from therecycled poollostoverthe2 loss andinsertionresults (±s.d.) obtainedbytheapproach showninA. AChRs. Scalebar, 20 synapses re-imaged todetermine theinsertionofnewlyrecycled musclewasthenbathed with freshsternomastoid strept-594, andthe again 2dayslater, andthelossofrecycled receptors determined.The recycled receptors; superficialsynapseswere imagedimmediately, and bathed withasaturatingdoseofstrept-594 tospecificallylabelthe later, muscle theanimalwasanaesthetizedandsternomastoid BTX-biotin followedbyasaturatingdoseofstrept-488. Three to4days small spotsoffluorescenceinthevicinityjunction(Fig.3B, postsynaptic membraneandininternalcompartmentsvisualizedas the AChRs labeledwithgreenstrept-488wereconcentratedinthe synapses wereimagedimmediatelyafterstrept-594(red)labeling, over thistimewerelabeledwithstrept-594.Whensuperficial biotin/strept-488, and3-4dayslaterthereceptorsthathadrecycled lapse imaging.Thesternomastoidmusclewas labeledwithBTX- accumulation ofbothgreenandredintracellularpunctausingtime- synapses; Fig.3A).Thispromptedustomonitortheformationand contained greenfluorescencefrompre-existing receptors( vesicles thatcontainedredfluorescencefromrecycled receptorsalso imaged 2ormoredayslater, weweresurprisedtofindthatallthe strept-594 (red).Whenrecycled andpre-existing AChRs werere- (green), and3-4dayslatertherecycled receptorswerelabeledwith sternomastoid musclewas labeledwithBTX-biotin/strept-488 same orindistinctintracellularvesicles. To examine this,the removal fromthepostsynapticmembrane,areinternalizedin 4488 recycled receptors. Fig. 2.Insertionofnewlyrecycled AChRsmatchestheremoval of RESEARCH ARTICLE ( A m. (B ) The sternomastoid musclewaslabeledwith ) Thesternomastoid ) Bargraphsummarizingtherecycled AChR n =50 Scale bars,20 stable vesiclescontainingfluorescent labelfrom pre-existing receptors. strept-594 labelsfrom recycled receptors are selectivelytargeted to the over thenext28hours(arrows), whereas othersare transient.The containing strept-488 from thepre-existing AChRsattime0are stable periodically overthenext28hours. Notethatanumberofthevesicles signal (arrowheads). ( existing AChRs(arrows), whereas someofthegreen spotslackred fluorescence from recycled AChRsalsohavegreen signalsfrom pre- recycled receptor labeling.Notethatallthevesiclescontaining red receptors. (A biotin/strept-488, then3dayslaterwithstrept-594 to labelrecycled AChRs. and targetedtovesiclescontainingstrept-488 from pre-existing Fig. 3.Fluorescent tagsfrom recycled receptors are endocytosed that thestreptavidin-Alexa tagshadindeed beenremoved and fluorescent vesicles weredevoid ofreceptors(Fig.4),suggesting 35 (pseudo-coloredblue).Interestingly, somegreenandred sectioned andimmunostainedwiththeanti-AChR antibodymAb the animalwas perfusedwithPFA andthemuscledissected, with strept-594(red)tolabelrecycled receptors.Two dayslater, with BTX-biotin/strept488(green),andthreedayslaterincubated investigate thispossibility, thesternomastoidmusclewas labeled expect atleastasubsetofthesevesicles tolackAChRs. To their receptorcomplexes andthensequestered,wewould derives fromstreptavidin-Alexa tagsthathave beenremoved from Discussion). had beenremoved frombothreceptorpoolsafterinternalization(see in theintracellularvesicles werefromstreptavidin-Alexa tagsthat biotin/strept-488 complexes, orthatthefluorescentpunctaobserved to stablevesicles alreadycontaininginternalizedAChR-BTX- removed recycled receptorsweredirectlyandspecificallytargeted (Fig. 3B,bottompanels).Theseresultsindicateeitherthatthe stable spotsthatcontainedbothstrept-488andstrept-594remained hours, althoughmany ofthegreenfluorescentspotshaddisappeared, stable spotsofgreenfluorescence(Fig.3B,middlepanels).After28 time point,faint redfluorescentspotsbegan toappear, but onlyatthe of thegreenspotsremainedwhereasothershaddisappeared.Atthis top panels).Whenthesamesynapsewas imaged8hourslater, some If theredandgreenfluorescenceobserved instablevesicles The sternomastoid musclewaslabeledfirstwithBTX- The sternomastoid ) Recycledandpre-existing AChRsimaged2daysafter m. B ) ANMJwaslabeledasinandthen imaged Development 133(22)

DEVELOPMENT AChRs werelabeledwithstrept-594, andsuperficialsynapses were and theanimalsallowed torecover. Three dayslater, therecycled denervated 6-8days earlierwerelabeledwithBTX-biotin/strept-488 have similar removal ratesindenervated muscle.Junctions Fig. 5C,D). innervated junctions (25%oforiginalfluorescence±7.1s.d.,n significantly lessthantheinsertionrateofrecycled receptorsin recovered atdenervated synapsesafter3days(Fig.5A,B),whichis Recycled AChRdynamicsinvivo found thatonly8%(±3.8s.d.,n muscle andthesamesynapseswerere-imaged.Interestingly, we initial labeling,new strept-594(red)was addedtothesternomastoid quantify thenumberofrecycled AChRs thathadbeeninsertedafter were locatedandre-imagedtomeasurethelossoffluorescence. To superficial synapseswereimagedand3dayslaterthesame then allbiotinsitesweresaturatedwithstrept-594(red). The earlier werelabeledwithasinglesaturatingdoseofBTX-biotin, and membrane. Innervated NMJsandjunctionsdenervated 6-8days affect ontheinsertionofrecycled AChRs intothepostsynaptic muscle denervation. We firsttestedwhetherdenervation hasany investigated whetherthislossrate couldbefurtheraffected by Given therapidremoval rateofrecycled AChRs, wenext recycled AChRdynamics Effect ofdenervationandmusclestimulationon tags removed (Fig.4). later stagesofrecycling thatalreadyhadtheirstreptavidin-Alexa indicating receptorsintheprocessofinsertion,or receptors only, withoutany fluorescentstreptavidin tags, recycling ordegradation. Athirdsubsetofpunctacontained possibly indicatingcomplexes thatwereintheprocessof contained fluorescentstreptavidin-Alexa tags andreceptors, sequestered, possiblyindegradative vesicles. Othervesicles whereas thosecorresponding tocyclingAChRs(whitearrows) maybemore dynamic.Scalebar, 20 Boxed areas (above)are enlargedbelow. Theresults suggestthatthevesiclescontainingfluorescent products (yellowarrows) bestable, may receptors contrast. andtheirstreptavidinColorsintheoverlayhavebeenadjustedPhotoshop(Adobe)tomaximize tagsafterinternalization. in laterstagesofrecycling and/ordegradation,ornewlysynthesizedreceptors intheprocess ofinsertion.Whitearrows vesiclescontaining indicate may correspondAChRs tointernalized streptavidin-Alexa tags;thesevesicles containing AChRs,butlackingany Blue arrows indicatevesicles correspond todegradativevesicles. whichprobablyinternalization, also recycled receptors after removed from pre-existing and of green andred streptavidin tags Yellow arrows indicateaccumulation correspond todegradativevesicles. whichmay after internalization, removed from pre-existing AChRs fluorescent streptavidin thathasbeen indicate accumulationofgreen with anti-AChR.Green arrows muscle sectionedandimmunostained with 2%PFA, thesternomastoid labeling, theanimalwasperfused Two daysafterrecycled receptor 594 (red) tolabelrecycled receptors. (green), then3dayslaterwithstrept- labeled withBTX-biotin/strept-488 muscle ofaliveanimalwasfirst labeled NMJ. Fig. 4.Confocalimagesofatriply Next, weinvestigated whetherrecycled andpre-existing AChRs The sternomastoid =20) oftheoriginalfluorescencehad =20; recycled receptors. that muscleactionpotentialsare sufficient toprevent thelossof fluorescence remaining±6s.d., prevented by thestimulationofdenervated muscles(94%of original original fluorescenceremaining ±5 s.d., AChRs observed indenervated musclesover the8hours(66%of to prevent asphyxia. Theresultwas dramatic:thelossofrecycled duration oftheexperiment, themousewas intubatedandventilated Hz for1secondevery 2secondsfortheentire 8hourperiod).For the at eitherendofthemuscle(3msecondbipolarpulses6-9V 10 action potentialswerethenelicitedviastimulatingelectrodesplaced label recycled AChRs. Superficialsynapseswereimagedandmuscle and then3dayslaterwas saturatedwithstrept-594tospecifically saturated (6-8daysafterdenervation) withBTX-biotin/strept-488, the postsynapticmembrane.Denervated sternomastoidmusclewas stimulation couldprevent therapidremoval ofrecycled AChRs from important forreceptorstability, wenext testedwhetherdirectmuscle doubling therateofremoval ofeachreceptor pool. similar effect onbothrecycled andpre-existing AChRs, nearly recycled receptorsfromthepostsynapticmembrane, andhasa indicate thatdenervation increasesthealreadyrapidremoval of of theiroriginalfluorescenceafter2days(Fig.6A,B).Theseresults than thehalf-lifeatinnervated synapses),andto43%(±6s.d., n=10) after1day(half-life~1.9days,alsonearlytwo timesfaster fluorescence frompre-existing receptorsdecreasedto70% (±6s.d., to 16%(±3s.d.,n at innervated synapses).Lossoffluorescencecontinued,decreasing of ~15hours,nearlytwiceasfast asthehalf-lifeofrecycled AChRs s.d., 5 (labeled recycled AChRs) decreased dramaticallytojust35%(± found thatafteronly1day, thestrept-594fluorescenceintensity imaged immediatelyandthenre-imaged12dayslater. We Because theabove experiments indicatethatmuscleactivity is n =17) oftheoriginalfluorescence(correspondingtoahalf-life =9) after2days.Atthesametime,strept-488 m. n =16) (Fig.7A-C).Thisindicates RESEARCH ARTICLE n =8) was almostcompletely 4489 n =9)

DEVELOPMENT synapses. graph summarizingmeasurements obtainedfrom manyinnervated of recycled AChRsreturnback tothepostsynapticmembrane.( innervated synapsetreated asinA,showingthatasignificantnumber intensity ±s.d.obtainedfrom manydenervatedjunctions.( scale. ( denervated muscle.Allthree panelsare displayedonthesameintensity indicating thatrecycling of AChRissignificantlydecreased in gained afterstrept-594 wasadded(6%oftheoriginalfluorescence), fluorescence waslostafter3days,whereas verylittle fluorescence was selectively labelrecycled AChRs.Notethat60%oftheoriginal to measure thelossoffluorescence, thenincubatedwith strept-594 to and imagedimmediately. Three dayslaterthesynapsewasre-imaged surface receptorswererestrictedtothejunctionalareaas 2002). Incontrasttountreatedcontrolmuscles,wheretherecycled phosphatases (Fletcheretal.,1993;Moult2006; with phenylarsine oxide(PAO), aknown inhibitoroftyrosine in receptorlocalizationatsynapseswhenthemusclewas treated to themembraneafterinitiallabeling.We foundastrikingalteration sternomastoid muscletolabelreceptorsthathadbeenrecycled back 9-hour incubationwiththedrug,strept-594(red)was addedtothe mice wereanesthetizedandventilated toprevent asphyxia.Aftera muscle thenbathedcontinuouslyinaphosphataseinhibitor. The muscle was labeledwithBTX-biotin/strept-488(green),andthe phosphorylation/dephosphorylation events. Thesternomastoid we next testedwhetherAChR recycling alsodependsupon (Ehlers, 2000;Estebanetal.,2003;Malinow and Malenka,2002), associated withpostsynapticreceptorcycling incentralsynapses Because phosphorylationanddephosphorylationevents are tyrosine phosphataseactivity Proper localizationofrecycled receptors requires 4490 AChRs. Fig. 5.Muscledenervationaffects thecontributionofrecycled B RESEARCH ARTICLE ) Bargraphsummarizingthemean percentage offluorescence ( A ) AdenervatedNMJwaslabeledwithBTX-biotin/strept-594 C ) An D ) Bar same proportion to~48hours.Scalebar:20 dropped to~15hours,and pre-existing AChRhalf-lifedecreased bythe life of~102hours.Indenervated synapses, recycled AChRhalf-life half-life of28hours,whereas pre-existing AChRshadanaveragehalf- day period.Ininnervatedsynapses, recycled AChRshadanaverage same synapsesasassessedbythe change influorescence overthe1- graph showingthelifetimeofrecycled andpre-existing AChRsfrom the receptor poolswere thenimaged,andre-imaged 1daylater. ( AChRs were labeledwithstrept-594. Fluorescence signalsfrom both first labeledwithBTX-biotin/strept-488, and3dayslaterrecycled pre-existing AChRsattheNMJ. Fig. 6.Denervationincreases theremoval ratesof recycled and Nine hourslater, whenthemusclewas labeledwithstrept-594, serine/threonine phosphatasesPP1andPP2A(Pahan etal.,1998). concentrations ofokadaicacid(10-100 labeled (asdescribedabove) andthenbathedindifferent in receptorrecycling, thesternomastoidmuscleoffourmicewas localization ofrecycled receptors. that tyrosinephosphataseactivity isinvolved inthecorrect localization ofpre-existing AChRs (Fig.8D).Theseresultsindicate recycled AChRs (Fig.8E,F),withoutaffecting thesynaptic Moult etal.,2006),causedthesameperi-synapticstainingof tyrosine phosphataseinhibitor, pervanadate (Madhavan etal.,2005; original synapse(Fig.8A).Treatment withanothercommon no pre-existing receptors(green)weredetectedoutsideofthe recycled receptorslabeledwithstrept-594(red),becauseat9hours presence ofredsignalintheperi-synapticregion isspecificto synaptic region aswellthejunctionalregion (Fig.8B,C).The original receptors,PAO treatmentresultedinredlabelingtheperi- To investigate thepotentialroleofserine/threoninephosphatases ( A ) AChRsatdenervatedNMJswere M), aspecificinhibitorof m. Development 133(22) B ) Bar

DEVELOPMENT loss offluorescence waslargelyabolished. ~66% offluorescence from recycled receptors remained indenervatedsynapses;however, whendenervated musclewaschronically this stimulated, recycled AChR stability. implies thatanunknown mechanismexists thatspecificallyalters (Burden, 1977;Missiasetal., 1996;Yumoto et al., 2005).This because innervated adultsynapsesonlyexpress theepsilonsubunit in thesubunit composition ofreceptors(gammaversus epsilon) it isunlikely thatthedifferences inlossratesareduetodifferences and epsilonsubunits of AChRs areabletoturnover atdifferent rates, et al.,1988).Althoughithasbeenshown previously thatthegamma NMJ activity isnottotallyblocked (Akaabouneetal.,1999;Lingle studies suggestthatlabeledreceptorsbehave normallyaslong binding itselfchangesthebehavior ofAChRs, anumberofprevious function. Althoughthereisnodirectevidence astowhetherBTX specifically andirreversibly bindstoAChRs, alsoblocksAChR BTX. However, itmustbeacknowledged thattheuseofBTX,which comparing theremoval ratesofreceptors that arebothtaggedwith obtained inourstudiesisduetoBTXbindingitself,aswe are membrane. Itisunlikely thatthedifference inreceptorlifetime though alloftheseAChRs areintermingled inthesamepostsynaptic receptors isfar shorterthanthatofthepre-existing AChRs, even AChRs atsynapticsites. dephosphorylation intheinsertionormaintenanceofrecycled work provides evidence forapossibleinvolvement oftyrosine stimulation completelyprevents receptorloss.Inaddition,this removal rateofrecycled AChRs, whereasdirectmuscle the numberofrecycled AChRs and increasesthealreadyrapid membrane. Theabsenceofmuscleactivity significantlydecreases synthesized AChRs thatarecolocalizedinthesamepostsynaptic AChRs turnover morerapidlythanpre-existing ornewly In thisstudy, wereportthatatfullyfunctionalsynapsesrecycled DISCUSSION activity was inhibitedandthemusclealsoshowed signsofswelling. 100 (>70%) was inhibited,whencomparedwithuntreatedmuscle.When okadaic acid,mostoftheserine/threoninephosphataseactivity phosphatase assaykit.Whenthemusclewas incubatedin10 the activity ofPP1andPP2Aphosphatasesusingaserine/threonine removing andhomogenizingthesternomastoidmuscle, andtesting drug invivo was testedattheend oftheimagingsessionby serine/threonine phosphatases(Fig.8G-I).Theeffectiveness ofthis that thetargeting ofrecycled receptorsdoesnotrequire specific stainingwas observed onlyinthejunctional zone,indicating Recycled AChRdynamicsinvivo presence (B recycled AChRsondenervatedmusclewere labeledwithstrept-594 andimagedimmediately, (A andthenagainafter8hoursintheabsence Fig. 7.Musclestimulationprevents lossofrecycled AChRsfrom denervatedNMJs. An interestingfindingofthiswork isthatthelifetimeofrecycled M okadaicacidwas used,allserine/threoninephosphatase ) ofstimulation.Scalebar, 20 m. (C ) Graphsummarizingthefluorescence measurements obtainedfrom manysynapses.After8hours, M possible thatduring thefirstweekofmuscle denervation, the affected withinthe firstweekofdenervation (datanotshown). Itis innervated synapses. However, theinhibitionofrecycling isnot depressed indenervated musclesynapseswhencomparedwith indicate thatthecontribution ofrecycled AChRs issignificantly of recycled AChRs inthepostsynapticmembrane.Ourresults NMJ orwiththeirmaintenanceatthepostsynapticmembrane. interferes withthedelivery ofinternalized receptorsbackintothe dissociation ofpre-existing receptors,but ratherthatitspecifically results suggestthattyrosinephosphataseinhibitiondoesnotcause distinguish betweenpre-existing andrecycled pools ofreceptors,our peri-synaptic region (DaiandPeng,1998).Becausewewereableto to thedissociationandmigrationofpre-existing receptorsintothe treated withtyrosinephosphataseinhibitors,whereiswas attributed made previously inthesynapsesoffrognerve/muscle co-cultures peri-junctional region. Interestingly, thesameobservation hasbeen increase theinsertionandaccumulationofrecycled receptorsatthe conceivable thattyrosinephosphatase inhibitorscouldselectively they have recycled intothepostsynapticmembrane.Itisalso mistargeting ofrecycled receptorsorincreasetheirmobilityonce associated proteinsattyrosinesitesduringtrafficking mightcausea NMJ. Thissuggeststhatthephosphorylationofreceptorsor boundary, whereaspre-existing receptorsremainintactwithinthe recycling, resultinginlabelingoutsideoftheusually sharp NMJ phosphatase activity cancauseselective disruptionofreceptor supported byourresults,whichshow thatinhibitionoftyrosine mechanisms maybeinvolved attheNMJ.Thispossibilityis Raymond etal.,1994;Roche1994);itispossiblethatsimilar regulate synapticstrength(Moultetal.,2006;Raymond etal.,1993; coordination ofphosphorylationanddephosphorylationevents may Wallace etal.,1991).Atcentralsynapses,ithasbeenshown thata activity (Fernsetal.,1996;Fuhrer1997;Wallace, 1994; removal andprevent receptorclustering,despiteongoingmuscle inhibition oftheseevents hasbeenshown toincreasereceptor involved inthestabilization ofAChRs (Caronietal.,1993),and For example, ithasbeensuggestedthatphosphorylationevents are affect theirstabilityonceinsertedintothepostsynapticmembrane. receptors withpostsynapticscaffolding proteins,whichinturnmay proteins mayoccur. Suchalterations mightaffect theinteractionof alteration ormodificationofAChR subunits orreceptor-associated trafficking (inearlyendosomesorspecializedrecycling endosomes), Muscle activity appears tobecrucialfortheinsertionandstability It ispossiblethatduringtheprocessofintracellularreceptor Three daysafterlabelingwithBTX-biotin/strept-488, RESEARCH ARTICLE ) and 4491

DEVELOPMENT synapse. Scalebars: 20 used instead,recycled receptors (red) were properly targetedtothe ( pre-existing receptors (green) remain restricted tothesynapse. that whereas recycled receptors (red) appearintheperi-synapticarea, tyrosineusing analternative phosphatase inhibitor, pervanadate.Note and contrastusingPhotoshop).( and recycled receptors (overlayimageswere adjustedforbrightness recycled receptors hadbecomemistargeted.( synaptic areas were labeled(arrows), indicatingthatsomeofthe strept-594 (red) todetermineifPAO affects AChRrecycling. Peri- hours andthenimaged.( continuously bathedinthetyrosine phosphataseinhibitorPAO for9 time 0andviewedathighdetectorgain9hourslater. Themusclewas mouse NMJpreviously saturatedwithBTX-biotin/strept-488 (green) at pool backtothepostsynapticmembrane.Interestingly, theabsence be attributed toadecreaseinthedelivery oftherecycled receptor in receptornumberatdenervated musclesynapses over timecould synapses (Akaabouneetal.,1999).Thissuggeststhatthedecrease neurotransmitter issufficient toprevent thelossofreceptorsfrom proposal, wehave previously shown thatspontaneousreleaseofa muscle denervation (Andreoseetal.,1995).Consistent withthis number ofsynapticreceptorsonlybegins todecline~9daysafter 1982; DennisandMiledi,1974),whichmightexplain whythe of acetylcholineneurotransmitterbySchwann cells(Brettetal., insertion ofrecycled receptorsismaintainedbyspontaneousrelease 4492 of recycled receptors intheperi-synapticmembrane. Fig. 8.Tyrosine phosphataseinhibitioncausestheaccumulation Whentheserine/threonine phosphatase inhibitorokadaicacidwas G-I) RESEARCH ARTICLE m. B ) Thejunctionwasthenincubatedwith D-F) Similarresults were obtained C ) Overlayofpre-existing ( A ) Liveadult Andreose, J.S.,Fumagalli,G.andLomo,T. new studytechniques. being degraded remainsunresolved andrequires thedevelopment of the questionofhow many timesareceptor canberecycled before may berecycled many times,losingtheirstreptavidin tageachtime, degradation pathway. Althoughthisresultsuggeststhatreceptors either intheprocessofrecycling orareatanearlystageinthe observed intime-lapseimaging;themoredynamicvesicles are sites ofdegradation, whichmaycorrespondtostablevesicles absence ofreceptorsinsomevesicles suggeststhatthese areindeed AChRs loseintracellularlywhileintheprocessofrecycling. The an accumulationoffluorescenttagthattherecycled and pre-existing than onceandsothestablefluorescentvesicles maycorrespond to being targeted fordegradation. Second,AChRs mightrecycle more products, implyingthatAChRs mightrecycle onlyoncebefore stable spotsoffluorescenceareparttheaccumulateddegradation existing AChRs. Inthisscheme,it istemptingtospeculatethatthe stable degradation vesicles containingpreviously internalizedpre- First, recycled AChRs couldhave atagthatdirectlyshuttlesthemto pre-existing receptors(seeFigs3,4).Thisraisestwo possibilities. vesicles withthepreviously internalizedgreenfluorescentlabelfrom streptavidin fromrecycled AChRs colocalizesinintracellular the recycled receptorpool.Anintriguingobservation isthatred existing poolmustentertherecycling pathway over timetomaintain degraded. Ifreceptorsrecycle onlyonce,thenafractionofthepre- receptors areabletorecycle backtothemembranebefore being pre-existing orpreviously recycled AChRs, orhow many times unclear whatproportionoftherecycled receptorpoolderives from continually insertedintothepostsynapticmembrane.However, itis type channelsaffects AChR stability(Rotzleretal.,1991). support ofthisidea,previous studieshave shown thatblockadeofL- that itcausescalciumreleasedirectlyfromintracellularstores.In calcium influxthrougheitherligand-gatedorL-typechannels, contraction causedbydirectstimulationcausesanincreasein AChR behavior isnotunderstood.Itpossiblethatmuscle neuromuscular transmissionormuscleactionpotentialsregulate 1989; RotzlerandBrenner, 1990).However, theway inwhich blocked endplates(Akaabouneetal.,1999;BrennerandRudin, the decreaseinAChR half-lifeobserved atsurgically denervated and synapses thataredenervated earlyindevelopment, andcanprevent reported toreversibly increasethemetabolicstabilityofreceptorsat Consistent withtheseresults,musclestimulationalonehasbeen stimulation alsoprevents theremoval ofrecycled receptors. (Bruneau etal.,2005),inthepresentstudywefoundthatmuscle denervated muscleprevents the removal ofpre-existing AChRs our previous findings,whichshowed thatthestimulationof loss, canstillbecontrolledbysynapticactivity. Inagreementwith membrane. Thisindicatesthatrecycled receptors,despitetheirrapid rapid removal ofthereceptorsthathave recycled backintothe removal rateofpre-existing AChRs, but alsoacceleratesthealready of muscleactivity following denervation notonlyincreasesthe Akaaboune, M.,Culican,S.Turney, S.G.andLichtman,J. W. REFERENCES Developmental StudiesHybridomaBank forproviding antibodies. Richard HumeandHansBrenner forhelpfuldiscussions.We alsothankthe Isabel MartinezandDavidSutterfortheirtechnicalhelpDrsJohnKuwada, Neurological Disorders andStroke Research GrantNS047332(M.A.).We thank This workwassupportedbytheUniversityofMichigan,NationalInstitute of Physiol. 483,397-406. acetylcholine receptors: control byneuralandmuscularinfluencesintherat. neuromuscular junctioninvivo. 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DEVELOPMENT