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SynapticTransmission Neuroscience201A PeterSargent 1. 2. 3. 4. Electricalvs.ChemicalSynapticTransmission CharacteristicsofLigand-GatedIonChannels Transmitter Release TransmitterReleaseStatistics “Non-traditional”methodsofmeasuringtransmitterrelease(voltage sensitivedyes,voltammetry,capacitance)– notcovered IntegrativeMechanisms– KevinBender SynapticPlasticity– RogerNicoll NeurotransmitterRelease/Uptake– RobEdwards SynapticModulation(transmittersactingviametabotropicreceptors)– JenniferWhistler Whatisasynapse? • Asynapseisasite ofcloseapposition betweenaneuronandatarget cell,whereanelectricalsignalinaneuronleadstoachangeinthe probabilitythatitstargetcellwillgiveanactionpotential. – iftheprobabilityincreases,thesynapseisexcitatory – iftheprobabilitydecreases,thesynapseisinhibitory Here,wearelookingatasectionofasynaptic boutonwithtwosynapticcontacts,ortwo synapses. tripartite synapse (pre,post,glia) tetrapartite synapse (pre,post,glia,ECM) (ECM=extracellular matrix) DityatevA,RusakovD (2011)Curr.Opin. Neurobiol.21:353-359. Electricalsynapses “gapjunction” = collectionofconnexons Electricalsynapsespermitdirectcurrentflowfrom onecelltotheother Smallmoleculescanreadilypassbetweencells connectedbygapjunctions(connexons) • Moleculesofupto~2kDcan passfromacelltoits electricallycoupled neighbors • Fluorescentmoleculessuch asluciferyellowwillpass fromcelltocoupledcell– suchcellsaresaidtobe “dye-coupled.” PatricioO'Donnell&AnthonyA.Grace,AlbanyMedical College,Albany,NYandUniversityofPittsburgh, Pittsburgh,PA Whyhavechemicalsynapses? Advantages Amplification Unlesstheterminalandthetargetcellarecomparableinsize, electricalsynapsesarenotlikelytobeeffective(impedance mismatch) Polarity(+,-) Modifiability/Plasticity Disadvantages Lossofreliability Speed(losetimeduetosynapticdelay)?? Howdoyoudistinguishanelectricalfroma chemicalsynapse? • Anatomy – dyecoupling? 1 • Physiology – synapticdelay? – quantalfluctuations? – reversalpotential? 2 Sequenceofevents(chemicalsynapses) 1. 2. 3. 4. 5. 6. 7. Anactionpotentialarrivesintheterminal, which activatesvoltage-dependentcalcium channels. Calciumenterstheterminalandpromotes fusionofsynapticvesicleswiththeplasma membrane. Transmitterisreleasedintothesynaptic cleft,and bindstoreceptorsonthepostsynaptic membrane. Thechannelsassociatedwiththese receptorsopen,whichallowionstoflow downtheirelectrochemicalgradient, excitingorinhibitingthecell. Transmitterisremovedfromthecleft,and thesynapseis“reset.” OUTLINE (wearegoingbackwards) 1. 2. 3. 4. Electricalvs.ChemicalSynapticTransmission CharacteristicsofLigand-GatedIonChannels TransmitterRelease TransmitterReleaseStatistics Simplestatefunctionfor anionotropicreceptor (skeletalmuscleacetylcholinereceptor– AChR) A+R « AR +A« A2R « A2R* « A2R closed Inside-out patch closed closed open desensitized (closed) bursting:movement betweenopen(A2R*)and closed(A2R)states A+R« AR+A« A2R« A2R*« A2R closedopendesensitized (closed) b A2RA2R* a closedopen forthemuscleAChR,a =700s-1andb =30,000s-1 Intheabsenceoftransmitter,therateofclosingwillbe governedbya.Thetimeconstantdescribingthefallin currentwillbe1/a,whichis1-2ms,whichoutlaststhe waveformof[ACh]whenacetylcholinesteraseispresent.The decaytimeofthecurrentthusreflectsthebehaviorofthe channels(notofthetransmitter,whichhasbeendestroyed). Atothersynapsesdecayofcurrentmaybeinfluencedby transmittersurvival. [transmitter],mM Whathappenswhentransmitterisavailablefora briefperiodoftime? 6 5 4 3 2 1 0 0 0.5 1.0 Time,ms 1.5 2.0 Principlesofrapid/focalsynaptictransmissionvia ionotropicreceptors • Theaffinityoftransmitterforreceptorsisusuallylow(10-100µM) • Releaseleadstoahighconcentrationoftransmitter(1-10mM)fora briefperiodoftimeinasmallvolume • Receptoroccupancycanbesubstantial,despitelowaffinityof receptorsfortransmitter(arereceptorssaturated?) • Diffusionanduptakeremovetransmitterfromthecleftquickly,so thattransmittertypicallyhasonly“onechance” tobindtoreceptor • Thesynapseiswelldesignedforrepeateduseathighfrequency(up to»1kHz) IonicBasisofExcitation/Inhibition • Excitation – Cation-selectivechannels (Na+ isthemajorchargecarrier) • ligand-gated:nicotinic,5-HT3,AMPA,NMDA,… • TRPchannels(transientreceptorpotential) • Inhibition – Anion-selectivechannels (Cl- isthemajorchargecarrier,HCO3- isalsoinvolved) • ligand-gated:glycine,GABAA,GABAC • Q:Canactivationofchloride-selectivechannelsbeexcitatory? – K+ selectivechannels Measuringtherelativepermeabilitiesofionotropic receptorchannels • Reversalpotential:thepotentialatwhichthecurrentis“reversed” in polarity,ornulled:thepotentialatwhichthereisnonetcurrent producedbyopeningthechannels • Whatisthereversalpotentialofalloftheleakchannelsofthecell, actingtogether? • Q: Howtomeasurethepermeabilityofachannel (orasetofchannels,actingtogether)? • A: Measureeffectofchangingioniccompositionuponreversal potential Thenicotinicacetylcholinereceptorchannelisa cationchannel replacemostofextracellular sodiumwithglucosamine (impermeant) AdamsDJ,DwyerTM,Hille B(1980)Thepermeabilityof endplatechannelstomonovalentanddivalentmetal cations.JGenPhysiol 75:493-510. Thenicotinicacetylcholinereceptorchannelisa cation channel(cont.) E1andE2representreversalpotentialsforNaandforthe ionX,respectively Einthisequationcandescribeonechannelorafamilyof channels(e.g.,allleakchannels). DoesAChopenonechannelortwo? FattandKatz(1951)andTakeuchi andTakeuchi(1960)established thatactivationofAChRsdrivesthe membranepotentialtoavalue near0mV. ThiscouldbeexplainedifACh openedonechannelclasspermeable tobothsodiumandpotassium,buta secondpossibilityisthatAChopens twodifferentchannels,oneforNa+ andoneforK+. TheGABAA channelisananionchannel here,inwardcurrent (negativeinsign,by convention)means movementofCl- in whichdirection? MouginotD,KombianSB,PittmanQJ(1998)ActivationofpresynapticGABAB receptorsinhibitsevoked IPSCsinratmagnocellularneuronsinvitro.JNeurophysiol79:1508-1517. UnderwhatconditionswouldmovementofCl- beexcitatory? Inhibitionoccursvia twomechanisms • Hyperpolarization – Whenthemembraneishyperpolarized,itismoredistantfrom thresholdand,generally,morecurrentisrequiredtoreach threshold • Shunting(short-circuiting) – Evenifthemembraneisnothyperpolarizedmuchbytheactionof aninhibitorytransmitter,theeffectisstillinhibitory,sinceCl- (or K+)movementwillopposetheconsequencesofinwardcurrent producedbytheactionofexcitatorytransmitters. – Shuntingisthepredominantformofinhibitioninthenervous system! OUTLINE 1. 2. 3. 4. Electricalvs.ChemicalSynapticTransmission CharacteristicsofLigand-GatedIonChannels TransmitterRelease TransmitterReleaseStatistics Calciumchannelsopeninresponseto depolarization Voltage-clamp • CalyxofHeldsynapse Isolatingcalciumcurrents pharmacologically • Sourcesofsynapticdelay YangY-M,WangL-Y(2006)Amplitudeandkineticsofactionpotential-evokedCa2+ currentandItsefficacyintriggering transmitterreleaseatthedevelopingcalyxofHeldsynapse.J.Neurosci.26:5698-5708. Calciumisnecessaryforrelease • ConsequencesofinjectingBAPTA,afast-actingCa2+ buffer BucurencluI,KulikA,SchwallerB,JonasP(2008)NanodomaincouplingbetweenCa2+channelsandCa2+ sensorspromotesfastandefficienttransmitterreleaseatacorticalGABAergicsynapse.Neuron57:536-545. Calciumissufficientforrelease • Consequencesofelevating[Ca2+]i artificially. Squidgiantsynapse DelaneyK,ZuckerRS(1990)CalciumreleasedbyphotolysisofDM-nitrophenstimulatestransmitterreleaseat squidgiantsynapse.JPhysiol426:473-498. Releaseisdependentonthe≅4th powerof[Ca2+]i CalyxofHeld(auditorybrainstem) SchneggenburgerR,NeherE(2000)Intracellularcalciumdependenceoftransmitterreleaseratesatafast centralsynapse.Nature406:889-893. Fateofreleasedtransmitter • Diffusion(Dis~0.5µm2/msec fortransmitters) – Diffusiontimeisdependentonthesquareofdistance(t≈x2/D) • Bindingtoreceptors • Removal[Uptake,Hydrolysis(ACh)] • Whathappensifyouinterferewithremovaloftransmitter? Distance (µm) Time (ms) (approx.) 0.1 0.01 1 1 10 100 100 10,000 1000 1,000,000 1 meter 32 years Directandindirectmethods ofmeasuringtransmitterrelease • Indirect – PostsynapticResponse – PresynapticResponse • Dyedestaining(e.g.,FM1-43) • Capacitancechanges • Direct – Voltammetry(amperometry) OUTLINE 1. 2. 3. 4. Electricalvs.ChemicalSynapticTransmission CharacteristicsofLigand-GatedIonChannels TransmitterRelease TransmitterReleaseStatistics Spontaneousrelease(“minis”) (followingfromtheworkofBernardKatzinthe1950s) 1. occurrandomly 2. occurintheabsenceof electricalactivity 3. causedbytherelease ofmulti-molecular packetsoftransmitter Evokedreleaseinlow[Ca2+]o shows“failures” (followingfromtheworkofBernardKatzinthe1950s) SaharaY,TakahashiT (2001)JPhysiol 536:189-197. Note“jitter” (asynchrony)inrelease process! Note fluctuationin responsesize. • Reductionof[Ca2+]oandorelevationof[Mg2+]oreducesEPSC sizeandleadsto“failures” ThesmallestEPSCsareequaltothesizeofthe spontaneouslyoccurringevents Note“jitter” (asynchrony)in releaseprocess! Notethatquantalresponses arenotuniform! IsaacsonJS,Walmsley B(1995)Countingquanta:directmeasurementsoftransmitterreleaseatacentral synapse.Neuron15:875-884. The“quantumhypothesis” (delCastilloandKatz,1954) • Transmitterisreleasedinmultimolecularpackets,or quanta.Thesepacketsarereleasedspontaneouslyatlow frequency.Thearrivalofanactionpotentialinthenerve terminalgreatlyincreasesthefrequencyofrelease.(from 1persecto~100permsec inthecaseofthefrognervemusclejunction) • Whatisthesignaldirectlyresponsibleforincreasingthe frequencyofrelease? TheKatzformalism • “Supposewehave,ateachnerve-musclejunction,a populationofN units capableofrespondingtoanerve impulse.Suppose,further,thattheaverageprobabilityof respondingisP,…,thenthemeannumberofunits respondingtooneimpulseism=N*P.” (delCastilloandKatz,J.Physiol.124:560-573(1954) • Whatisthephysicalidentityoftheunits? • foreachmemberofthepopulationN,theremayormay notareleaseeventinresponsetothearrivalofanaction potential • transmitterreleaseisaprobabilistic,orstochastic,event.It canbestudiedusingstatistics. Howtotestthe“quantumhypothesis?” (doestransmitterreleasereallyoperatethisway?) • Trytopredicttheaveragenumberofquantathatare released,assumingthatthehypothesisiscorrect,and comparethistoadirectmeasurement ofthe“quantum content.” • Thedirectmeasurementofquantalcontentisthenumber ofquanta.Usually,however,youcannotcountquanta. Instead,youdividetheaverageamplitudeoftheevoked response(epp)bytheaverageamplitudeofthemepp (assuminglinearsummation). – IsitOKtoassumelinearsummation? Usethebinomialdistributiontopredictthequantal content • Thebinomialtheoremdescribesdistributionsofoutcomes acrossasetoftrialswherethereareonlytwopossible outcomes(heads/tails,success/failure,release/norelease)per trialpermemberofthepopulationN. • Ifp=probabilityofsuccesspertrial,thentheprobabilityP (caps!)ofgettingksuccessesinntrialsis ö ænö æ n! ænö k ÷÷ çç ÷÷ = çç P(k ; n, p) = çç ÷÷ p (1 - p) ( n -k ) ,where è k ø è k!(n - k )!ø k è ø • Theaveragenumberofsuccessesism=N*p • Problem!– whatifyoudon’tknowNorp? “nchoosek” UsingthePoissondistributiontopredict quantalcontent (see“binomial”handout) • Poissonisaspecialcaseofthebinomial,wherep<<1and n® ¥ m k -m P(k ; n, p) = k! e Youdon’thavetoknowp!!! • Probabilityofgetting0successes(afailure) m0 -m -m P(0; n, p) = e =e 0! T P(0; n, p) = 0 ,whereTisthenumberoftrialsandT0 isthenumberoftrials T resultinginafailure æT ö m = ln çç ÷÷ the“failuresmethod” è T0 ø (later,theCVmethod(m=1/CV2) SynapsesobeyPoissonstatisticsatlowprobabilities ofrelease mbythe“failuresmethod” mbythe “direct method” IsaacsonJS,WalmsleyB(1995)Countingquanta: directmeasurementsoftransmitterreleaseata centralsynapse.Neuron15:875-884. • Measurem(directmethod)andcomparetomcalculated fromthefailuresmethod Thequantumhypothesisisverified. Sowhat? • Whatisthesignificanceofthefactthatthepoisson distributionsuccessfullypredictsm? • Biology – thatevokedresponsesareconstructedfromthesame“units” thatoccurspontaneously(thisiscontroversial– e.g.,Kavalali’s work) – thatthese“units” (releasablequanta)arereleasedindependently • Methodology – youmightbeabletoestimatewhetherachangeinthesizeofan EPSCiscausedbyachangeinquantalcontent “Synchronous” vs.asynchronousrelease Releaseisnevertrulysynchronous, SaharaY,TakahashiT (2001)JPhysiol 536:189197. Note“jitter” (asynchrony)in releaseprocess! but itcanbeclosetosynchronous(esp.atphysiologictemperature). SargentPB,Saviane C,NielsenTA,DiGregorio DA,SilverRA(2005) J.Neurosci.25:8173-8187. Asynchronous(Delayed)Release AtluriPP,RegehrWG(1998)Delayedreleaseofneurotransmitterfromcerebellargranulecells.JNeurosci18:8214-8227. IntroducingaCa2+ bufferintotheterminalreduces delayedrelease • parallelfiber– Purkinjecell synapseincerebellum • EGTA-AMintothebath EGTA-AM iCa PSC PSC esterase PSC EGTA ChenC,RegeherWG(1999)Contributionsofresidualcalciumtofastsynaptictransmission.JNeurosci19:6257-6266. Synapticconnectionsshowwidediversityinthe importanceofasynchronousrelease HefftS,JonasP(2005)AsynchronousGABAreleasegenerateslong-lastinginhibitionatahippocampalinterneuron–principal neuronsynapse.NatureNeurosci.8:1319-1328. StructureoftheNerveTerminal • synapticvesicles • “docked” vesicles • activezones(AZs) • postsynapticdensities (PSDs) fromHeuserlabwebsite Thevesiclehypothesis Aquantumoftransmitteristhatamountstoredinasynapticvesicle. Releaseoccursvia exocytosis. • Stimulationproducesexocytoticevents,hereseenaspitsontheCfaceofthe plasmamembraneinfreezefracture. HeuserJE,ReeseTS,DennisMJ,JanL,JanYN,EvansL(1979)Synapticvesicleexocytosiscapturedbyquick freezingandcorrelatedwithquantaltransmitterrelease.J.CellBiol,81:275-300. Thevesiclehypothesis Aquantumoftransmitteristhatamountstoredinasynapticvesicle. Releaseoccursvia exocytosis. • Capacitancemeasurementsshowthatreleaseofaquantumof transmitterisaccompaniedbyanincreaseinthesurfaceareaofthe terminalbythesurfaceareaequivalenttoa~50nmsphericalvesicle. 160,776 SunJ-Y,WuX-S,WuJ-G(2002)Singleandmultiplevesiclefusioninducedifferent ratesofendocytosisatacentralsynapse.Nature417:555-559. 320,541 Nerveterminalscontaindistinctpoolsofvesicles • • • WhatisthephysicalcorrelateofN,thenumberof readilyreleasablevesicles? N<<#vesicles N@ numberofanatomicallydockedvesicles(very approximately!) readilyreleasablepool- RRP RizzoliSO,BetzWJ(2005)Synapticvesicle pools.NatRevNeurosci6:57-69. SchneggenburgerR,SakabaT,NeherE(2002)TrendsNeurosci 25:206-212. Remainingcontroversies(partiallist!) • Isexocytosisaccompaniedby“fullcollapsefusion” orthe vesicle,orbya“kissandrun” mechanism? – – • • Capacitancemeasurementssuggestthat“kissandrun” maynotbeprevalent.(Whatkindofdatawouldsupport “kissandrun?”) “Kissandrun” and“fullcollapsefusion” maybothoccur naturallyatdifferenttimes,dependingonthestateofthe terminal. Arespontaneouslyoccurringevents(“mEPSCs”)identical tothebuildingblocksofEPSCs? Howisendocytosislinkedtoexocytosis? Poissonandbinomialdistributions • Isthepoissondistributionrelevantwhensynapses operateatphysiologicalcalcium? • NO!!! – Pisnolongersmall! – Atmanysynapses,Nisnotlarge,andsothePoissonwouldnot apply,strictly,evenif[Ca2+]o werereduced. – Note,however,thatinpractice,itdoeswork,aslongaspissmall. • FormostCNSsynapsesatphysiological[Ca2+]o,the binomialdistribution,notthepoissonone,shouldbeused toanalyzetransmitterrelease. • Topredictthedistributionofoutcomesusingthe binomial,weneedtoknowmorethansimplym,alas. Additionalchallengestostudyingthestatisticsof transmitterreleaseintheCNS 1. Inmanycells,youcannotrecordsynaptic responsesfaithfullyfromthecellbody(space clamperrors). 2. Mostcentralneuronsaremultiply innervated.Youmaybeabletorecord evokedresponsesfromoneinput,butyou cannoteasilystudyspontaneousreleaseonly fromthatinput.(convergence) 3. SimplebinomialmodelsassumethatPis uniformacrossthepopulationN,butit’snot. (non-uniformityofP) 4. SimplebinomialmodelsassumethatQis uniform,butit’snot.(non-uniformityofQ) Measuringquantalparameterswithoutknowingabout mEPSCs:variance-meananalysis • Measurethemeanandthevarianceofsetsofresponses tostimulationofasingleinputunderdifferent probabilitiesofrelease. • Youdonotneedtomeasurespontaneousevents, • Fromthebinomialdistribution,thevarianceandmeanare relatedaccordingto: s2 =QI– I2/N where I=thecurrent, Q=quantalsize,meaningthe responsetoonequantum,and N=thenumberofreleasablequanta Fromthebinomial 1. Mean=NPorNPQ 2. s2 =NP(1-P)orNPQ2(1-P) SubstituteforP EstimatingN,P,andQ(cont.) σ2 =QI– I2/N y=Ax– Bx2 ClementsJD,SilverRA(2000)Unveilingsynapticplasticity:anewgraphicaland analyticalapproach.TrendsNeurosci 23:105-113. Note:thisisthesimplebinomial versionoftheexpression,butthis analysiscanreadilybemorphedintoonethataccountsfornonuniformityinPandinQ(themultinomial). IsQindependentofP? • Binomial-basedanalysesoftransmitterreleaseassume thatthequantalresponse,Q,isindependentofP – i.e.,thattheresponsetoeachquantumisthesame,regardlessof howmanyquantaarereleased • Isthisagoodassumption?Aresynapses“linear?” (i.e., doesthepostsynapticcellprovidealinearreadoutofthe numberofquantareleased?) Aresynapses“linear?” (cont.) (IsQindependentofP?) • Thesuitabilityofthepoisson statisticssuggeststhat releaseoccursindependentlyformembersofthe populationN(presynaptic). • Butwhatabouttheconsequences ofrelease?Isthe responsetoreleaseofonequantumindependentofthe releaseofanother?(postsynaptic) 1. 2. – Whatisthespatialrelationshipbetweenseparatereleasesites thatconstituteasynapticconnection? Canmorethanonequantumoftransmitterbereleasedper activezone? Ifreleasesitesarewellseparated,andifnomorethanone quantumcanbereleasedperactionpotentialpersite,thenthe postsynapticcellshouldbeabletosumtheresponsesto individualquantalinearly. UnivesicularRelease SilverRA,LubkeJ,Sakmann B,FeldmeyerD(2003) Science302:1981-1984. • MeasureN(variance-meananalysis)and measure#boutons. • N=#boutons. • Releaseisunivesicular,andsincerelease sitesarewidelydistributed,theresponse shouldscalelinearlywiththenumberof quantareleased Somaanddendritesofpresynapticcellareinred Axonofpresynapticcellisinblue Somaanddendritesofpostsynapticcellareinblack Axonofpostsynapticcellisingreen, MultivesicularRelease BiroAA,HolderithNB,NusserZ (2006)JNeurosci.26:1248712496. • MeasureN (variance-mean analysis)and measure#boutons. • N>#boutons. Furtherevidenceformultivesicularrelease Climbingfiber-Purkinjecell(CF-PC) • AsPincreases,mwillincrease synapseincerebellum • Whathappenstopeak[glu]? • Lookateffectsofslow-offandfast-offantagonists NBQX isahighaffinity competitiveAMPAR antagonist.Itsefficacy willnotdependupon [glu]. g-DGG isalowaffinity, competitiveAMPAR antagonist.Itsefficacy willdependupon[glu]. Showing EPSC before andafter g-DGG WadicheJI,JahrCE(2001)Multivesicularreleaseatclimbingfiber-Purkinjecellsynapses. Neuron32:301-313. Furtherevidenceformultivesicularrelease (cont.) ClimbingfiberPurkinjecell synapsein cerebellum Pairedpulsedepression PPRisdifferentincontrol andinthepresenceofgDGG.It’snotdifferentin NBQX. PPR(control)=0.45 PPR(g-DGG)=0.25 What’sgoingon? Depressioniscausedin partbydepletionof vesicles.Fewerquantaare releasedinresponsetothe secondstimulus.Thedata suggestthatpeak[glu]is largerinresponsetothe firstpulse(g-DGGisless effective).Thisagainis evidenceofmultivesicular release. PPR(control)=0.50 PPR(NBQX)=0.50 WadicheJI,JahrCE (2001)Multivesicular releaseatclimbing fiber-Purkinjecell synapses.Neuron 32:301-313. Q(quantalsize)isnotuniform CV is0.2-0.6 (CV=S.D./mean) • Intrasite/intersitevariability • Sourcesofvariability – intrasite IsaacsonandWalmsley(1995) • amountofglutamateinvesicle? • isalloftheglutamateinthevesiclereleased, andreleasedrapidly?(areallexocytotic eventssimilar?) • probabilisticnatureofevents(Popen forreceptor’schannel) – intersite • isthedensity/numberofglutamatereceptors(acrosssites)similar? • isthedensity/numberofglutamatetransporters(acrosssites)similar? • isthegeometryofeachsynapticcontactsimilar? OUTLINE 1. 2. 3. 4. 5. Electricalvs.ChemicalSynapticTransmission CharacteristicsofLigand-GatedIonChannels Transmitter Release TransmitterReleaseStatistics SynapticPlasticity(iftime) ICING!!! SynapticStrength I=m*Q=N*P*Q I=response,measuredtypicallyincurrent(e.g.,peakofEPSC) m=avg.numberofquantareleased(=N*P) N=numberofreleasablequanta[readilyreleasablepool(RRP)of vesicles] P=averageprobabilityofrelease,and Q=quantalresponse:thesizeoftheresponseofthepostsynaptic celltoonequantumoftransmitter Note:thisassumesthatthesystemislinear! Synapticstrengthisdependentuponuse(upon history) • Facilitation neuromuscular junction(lowCa2+) • Paired-pulse facilitation 4 pA mossyfiber– granule synapseincerebellum 2 msec Ispaired-pulsefacilitation(PPF) presynapticorpostsynaptic? • Synapticstrength:I=N*P*Q – Nisgenerallyassociated withpresynapticfactors – Pisgenerallyassociated withpresynapticfactors N*P=m(numberofquanta) – Qisgenerallyassociated withpostsynapticfactors • Howtoresolvethequestion? – measurem,fromthefailuresmethod m=ln(T/T0) IsaacsonJS,WalmsleyB (1995)Countingquanta: directmeasurementsof transmitterreleaseata centralsynapse.Neuron 15:875-884. Whatcausesanincreaseinm forthesecondoftwo stimuli? • Largeractionpotentials? – butaren’tactionpotentialsallornone? • Morecalciumenteringduringtheactionpotential? – iCa canbelargerforthesecondoftwoAPs – however,facilitationcanoccureveniCa isunchanged. • Residualcalciumhypothesis: Nerveterminalsfacilitatebecausesome ofthecalciumthatenterstheterminalduringthefirstaction potentialisstillpresentwhenthesecondimpulsearrives.This “residualcalcium” isresponsibleforgreaterreleaseafterthesecond impulse. – Thisisunlikelytobetheentireexplanation,sincetheamountof“residualcalcium” measurableisnotsufficienttoexplainthedegreeoffacilitation. • Calciumbuffersaturationhypothesis (relatedtotheresidualcalcium hypothesis).Calciumbuffersarepartiallysaturatedasaresultofthe calciumthatenterstheterminalinresponsetothefirstimpulse,and lesscalciumissequesteredfollowingthesecondimpulse. Interplaybetweenfacilitationanddepression 1.25 mM Ca2+, 3.00 mM Mg2+ 4 pA 2 msec 3.00 mM Ca2+, 0.75 mM Mg2+ 20 pA granule cell mossy fiber 2 msec SargentPB,NielsenT,DiGregorioDA,SilverRA(unpublished) Theprobabilityofreleaseisdependent upontwofactors • Ifthereareanatomicallydefinablesitesfromwhich transmitterisreleased,thenPwilldependbothon whetherthesiteisoccupiedwithavesicleandwhether,if occupied,thevesiclefuseswiththeplasmamembrane • P=Poccupied *Pfusion=Po *Pf • Ifthesiteisnotoccupiedwitha“release-ready” vesicle, thentherewillbenoreleaseeventthere. Whydoesasynapsethatfacilitatesatlow[Ca2+]o depressathigh[Ca2+]o? • Depression(someforms) iscausedbythefailureofthe nerveterminaltoreplacevesiclesreleasedduringthefirst response.Thisprocesstakestime– generallytensofms. • Recallthatm=N*P=N*Po *Pf – “residualcalcium” willincreasePf – depletionofvesicleswilldecreasePo – ifPf increasesmorethanPo decreases,wewillhavefacilitation. Butwhen[Ca2+]o ishigh,therewillbemuchdepletioninresponse tothefirstpulse,andPo willdecreasebymorethanPf increases, resultingindepression. Synapsesvaryintheirresponse totrainsofstimuli Climbingfiber– Purkinjecell (cerebellum) Phigh depressionduringtrain Parallelfiber– Purkinjecell (cerebellum) Plow facilitationduringtrain DittmanJS,KreitzerAC,RegehrWG(2000)Interplaybetween facilitation,depression,andresidualcalciumatthreepresynaptic terminals.JNeurosci20:1374-1385. Longer-lastingformsofplasticity Facilitation Augmentation PTP(post-tetanicpotentiation) LTP/LTD fin!