1SynapseEliminationinthePNS

Wehavepreviouslydiscussedthedetailsofeventsthatoccurtodirectthe formationandspecializationoftheNMJasamodelsynapse. Duringdevelopmentalsynapseformation,motoneuronswillinnervateany musclefiberthattheyencounterintheirtargetenvironment. Selectivityofindividualtargetcellsisnotasdifficultherebecausemuscle fiberswithdifferentfunctionsarenaturallywithindifferentmusclessome distanceaway. Therefore,axonguidancemechanismsmightbeamongthemost importantinbringselectiveaxonstowardspecificmuscletarget environments,butafterthatpoint,thereisnogreatneedtobeselective amongindividualmusclefibers. Inthebrain,thechoiceofpostsynapticpartnersismorecomplex,and probablymoreprecise. SynapseeliminationinthePNS Followingaxonguidanceandtheformationofselectivesynapses,the processofadaptationandadjustment(marriageordivorce)takesplace. IfonefollowstheformationofsynapsesintheNMJ>initiallymorethan oneaxoninnervateseachnerveterminalareaonthemuscle....thenthisis reducedtooneaxon/muscle.... Isthisaprocessofeliminationofsynapsesthatinitiallyformedon inappropriatetargetmuscles....?NO >notinappropriatemuscle,butitisinappropriatetohavetwo axonsoneachmuscle..... Theprocessofaxonguidanceandselectivityofsynapticconnections avoidstheproblemofinappropriateconnectionsbeingformedduring developmentintheneuromuscularsystem......ItisageneralPRINCIPLE

thatneuronsformappropriateconnectionsduringdevelopment. However,thisisonlyaqualitativematchingofneuronswiththeir appropriatetargets.....asecondphaseofrearrangementisneededtocreate theprecisequantitativerelationshipsbetweenneuronsandtheirtargets. Inotherwords,eachaxonmustinnervatetheappropriatenumberoftarget cells,andeachtargetmustreceiveinnervationfromtheappropriate numberofaxons. Ingeneralthisinvolvesthelossofconnectionsthatwereestablishedearly indevelopment........Butnottheresultofneuronaldeath. Noterrorcorrectionorrandomlossofconnections...anactiveprocess thoughttobeduetoanexchangeofinformationbetweensynapseand targetcell. Thegeneralprinciplethatthesesystemsdemonstrateisthatthis rearrangementoccursthroughcompetition.Thatis,competitionbetween axonsforsinglesynapticsitesonidentifiedtargetcells. AttheNMJ: HereasingleMNwillsynapsewithmanymusclefiberswithinasingle muscle,butusually,eachmusclefiberonlyhasonesynapse. Ofcourseitdoesntstartoutthatway.....Althoughsinglesynapticsitesare supportedonmusclefibers,severalaxonscanpopulatethatsiteinitially. Asdevelopmentproceeds,graduallythenumberofaxonsinnervatingeach synapticsitedropstoone. Thisrearrangementisoftencalledsynapseelimination. Thissynapseeliminationdoesnotresultinthedeathofneurons,asaxons retreatfromonlysomeoftheirsynapticsites.....ratheritisareductionin thesizeoftheMotorUnit:Thenumberofmusclecellsthatone motoneuroninnervates. Historically,thishasbeenexaminedprimarilyusingtwoapproaches:

Silverstaining,andelectrophysiologybythresholdstimulation...recently,a combinationofgenetictoolsandfluorescentmicroscopyhasbeenuseful.

UsingrepeatedobservationsofanidentifiedNMJlabeledwithfluorescent markersonecanobservethecompetitionoftenonesynapsetakesover thesynapticsiteasanothervacates(oriseliminated). Althoughyoucanhavesynapseeliminationwithouttakeover Theroleofactivityinsynapseelimination Activitydependentcompetition: Doesactivityplayarole? Exp:Paralyzethenervewithalocalanesthetic....synapseelimination slowsdown. Conversely,whenanincreaseinactivityisimposed...synapseelimination speedsup. Also,theoverallamountofstimulationmaynotbeasimportantasthe patternofactivity. CulturedNMJexperiment....lookfortwosynapsesonasinglemusclefiber invitro.Thenstimulatethemeithersynchronously,orasynchronouslyat thesamerate........ Whenstimulatedtogether,thesynapsesdidnotchange....whenstimulated asynchronously,oneofthetwoshowedadramaticdecreaseintransmitter released.....Calledheterosynapticcompetition. Thepatternofactivityseemstobeimportant. Therehasbeenonegeneralhypothesisaroundwhicheveryoneseemsto structuretheirexplanationsofthisactivitypatternobservation:

HEBBsrule(D.O.Hebb,1949): WhenanaxonofcellAisnearenoughtoexciteacellBandrepeatedly andpersistentlytakespartinfiringit,somegrowthormetabolicchange takesplaceinoneorbothcellssuchthatAsefficiency,asoneofthecells firingB,isincreased. Terminalsarereinforcedwhentheyareactiveatthesametimeasthe targetcell.Cellsthatfiretogether,wiretogether Onecleardifferencebetweensynapsesarisingfromdifferentneurons, mightbethatdifferentneuronswillfireactionpotentialsatdifferenttimes, whereasdifferentsynapsesfromthesameaxonwillallfireaction potentialsatthesametime. Invivoobservation: Interestingly,ifyourecordtheactivityofmusclenerves,theystartout synchronized,butthenafterbirth,theybecomeunsynchronizedthis correspondstothebeginningofsynapseelimination. Limitationsonmechanisms? Thisactivitydependentprocessappearstorequirethatsynapsesare relativelyclosetooneanother. TheNMJisconsiderednotonlyasimplesystem,butanextremecase whereactivityinasinglesynapsecancausethemuscletoreachthreshold andfireanactionpotential.....herecompetitionwouldbeexpectedtobe strongestandmightstretchthewholelengthofthepostsynapticcellbut itdoesnot. .....onsomeverylargemusclefibers,morethanonesynapseissupported. Ontheselongmusclecells,twosynapsescanbetoleratediftheyaremore thanonemillimeterfromoneanother. Here,competitionisapparentlyreducedduetodistance......

InfactthatinvitroexperimentwetalkedaboutinculturedNMJsdoesnot workifthesynapsesarefarapartonthemusclecell. Whenthistypeofcompetitiondoeswork,thereasonthatthesynaptic transmissionbecomesweakerinonesynapseisthatpostsynapticreceptors areremovedfirst ThisreceptorchangeappearstoalsoworkattheNMJinvivo.... Aninterestingexperimentthatisonlypossiblewiththediscoveryofvital dyesthatselectivelylabelthelivingsynapsewithoutalteringitsstructure orfunction(4Di2Asp,orgeneticexpressionofGFP).Thereareafew musclefibersinvivothataresuperficialandamenabletoperturbationand repeatedobservation(sternomastoidmuscleinmice). Exp:usetheadultNMJasamodelforwhatmighthappeninthe developingsystem...alteractivitybyblockingpostsynapticreceptors Theprocesseswehavejusttalkedaboutpredictsthatifyoupreventone releasesiteofaNMJfromactivatingthepostsynapticcell,thatreleasesite shouldbeeliminated. BaliceGordonandLichtmanhavedonethisbyfocallyapplyinga postsynapticreceptorblockerjustbeneathasmallsubsetofthereleasesites onanadultNMJ>Thosesynapsesareessentiallynowsilent;sewthe animalbackupandlethimusethatsynapse..... Overthesubsequentfewdaysandweeksinvivo(asthissynapsewasused bytheanimal),thesamesynapsecouldbereexaminedattwoday intervalsandchangesobserved.... 1)Theblockedreleasesitesretracted....! 2)Ifthewholeterminalwasblocked>nochangeinterminal morphology. Theseobservationssuggestthehypothesisthatactivemusclecanrelease

asignalthatcaneliminatenerveterminalsthatarenotalsoactive...AND thatanactivesynapseisprotectedfromthiseliminationsignal... Ithasbeenproposedthatkinaseactivationpostsynapticallycanalter receptornumber.

WhatmechanismsgovernsynapseeliminationattheNMJ? Severallinesofevidencesupporttheideathataxonscompeteforeach synapticsite.....theloserswithdrawfromthatsitebecausetheyhavebeen lesssuccessfulinacompetitionforsomething. Thisideaissupportedbyobservationsinsomemusclesinwhichthe experimenterhasmadeiteasierforsomecells......cutoutmostofthe competition.Whendonewithmotorunitsthatnormallydecreaseinsize, nowtheydont. Butwhataretheycompetingfor? Mostinvestigatorsfeeltheyarecompetingforsometypeoftrophic support...somemoleculemadebythetargetcell,andsecretedbythe target,thatsupportsthemaintenanceofthesynapse. Infact,recentworkshowedthatifyouinjectaproteinsynthesisinhibitor intoonepostsynapticmusclecellthatcellstopsmakingprotein,andthe nerveterminalonthatcellretracts! Maybeduringdevelopment,themusclemakesandsuppliessomething basedonactivitythatsupportsonlyonenerveterminal. Experimentalworkhasarguedthatitisnotalimitintheamountoftrophic molecules,butatimingissue.....alimitin when thesemoleculesare provided.

Observationsofthetimecourseofsynapseeliminationfromthese experimentshaveshownthattheAChRareremovedfromunderasynapse beforethatsynapseisremoved.... Therefore,bothaprotectivesignalandaneliminationsignalare hypothesizedtobetriggeredbypostsynapticreceptors,thesesomehow regulatethepresenceofthenerveterminal.... Thereleaseoftrophicfactorsbythetargetcell,andtheabilityofsynapses tosensethesefactorsappearstobeactivitydependent. Therefore,thetwocellsmustbeactiveatthesametimeforthereleased substancetobesensedbythenerveterminal. Oneideaisthatactivereceptorsgenerateasignalinthepostsynapticcell thatcandiffusebacktothenerveterminaltoinfluencestabilizationofthe synapse. Pooexperiment.....Newanduntestedhypothesisthatisattractive .....postsynapticmuscleactivitygeneratescalciumentrythroughAch receptors,whichtriggersthegenerationofNitricoxide....agaswithavery short1/2lifethatcandiffusebacktothenerveterminal. Infact,inhisexperiments,thetwosynapsesdidnothavetobeactive simultaneously,aslongastheywereactivewithin10msecofoneanother. IfN.O.wasreleased,thepropertiesofthisgaswouldfittheseobservations. Exptest: BlockN.O.withhemoglobin>blocktheheterosynapticcompetitionin vitro. N.O.isoneofthenewestneuronalregulatorsdiscovered...ithaspotent stimulatorypropertiesforgeneratingcGMPincells....andtherefore,could workherebystimulatingasecondmessengercascadeinnerveterminals thatcouldonlybeactivatedifthesynapsewasactive.

Thistypeoflocalsynapticsignalingcouldexplainhowatargetcellthat waseliminatingoneofitsaxons,couldallowtheremainingaxonto enlarge Morerecently(Jeetal.,JournalofNeuroscience33:9957)June,2013 Anewcandidatefortheprotectionandeliminationsignalswas presented. Theneedforpostsynapticmuscleactivationisstillinplace,butnowthe calciumentrythroughpostsynapticreceptorstriggersthereleaseofa growthfactorprecursor:proBDNF DependingonifthisprecursorisprocessedintoBDNFornot,itcanactsas arewardorpunishment. ProBDNFbindstothep75receptorwhichcausesretractionofnerve terminals BDNFbindstotheTrkBreceptorwhichcausessynapticmaturationand increasesinsynapticstrength. ThekeyisregulationofcleavageofproBDNFwhennerveterminalsare active,theysecretemetalloproteaseswhichconvertproBDNFintoBDNF. Therefore,ifthepostsynapticcellisactive,butthepresynapticnerve terminalisnotpunishmentsignalcausedbyproBDNFbindingtothe nerveterminalp75receptor. Incontrast,ifthepostsynapticcellandthepresynapticcellarebothactive, themusclereleasedproBDNFisconvertedintoBDNFandisnotonly protective,butsupportiveatthatnerveterminal. Inoneoftheirkeyexperiments,theseinvestigatorsshowedthatinjectionof ablockingantibodytop75preventedsynapseelimination,whileinhibition ofTrkBreceptorsacceleratedsynapseelimination.