The Active and Periactive Zone Organization and the Functional Properties of Small and Large Synapses

PERSPECTIVE published: 24 May 2016 doi: 10.3389/ nsyn.2016.00012 The Ac i e and Pe iac i e Zone O ganiza ion and he Func ional P ope ies o Small and La ge Synapses Raquel Cano and Lucia Taba es* Depa men o Medical Physiology and Biophysics, School o Medicine, Uni e si y o Se ille, Se ille, Spain Edi ed by: Ma ín Camma o a, Fede al Uni e si y o Rio G ande do No e, B azil Re iewed by: Je ey Sco Diamond, Na ional Ins i u es o Heal h, USA Michele H. Jacob, Tu s Uni e si y, USA *Co espondence: Lucia Taba es [email p o ec ed] Recei ed: 21 Ma ch 2016 Accep ed: 09 May 2016 Published: 24 May 2016 Ci a ion: Cano R and Taba es L (2016) The Ac i e and Pe iac i e Zone O ganiza ion and he Func ional P ope ies o Small and La ge Synapses. F on . Synap ic Neu osci. 8:12. doi: 10.3389/ nsyn.2016.00012 The a i al o an ac ion po en ial (AP) a a synap ic e minal elici s highly synch onized quan a elease. Repe i i e APs p oduce successi e synap ic esicle (SV) usions ha equi e managemen o spen SV componen s in he p esynap ic memb ane wi h minimum dis u bance o he sec e o y appa a us. To his end, he synap ic machine y is s uc u ed acco dingly o he s eng h and he ange o equencies a which each pa icula synapse ope a es. This esul s in a ia ions in he numbe and dimension o Ac i e Zones (AZs), amoun and dis ibu ion o SVs, and p obably, in he p ima y endocy ic mechanisms hey use. Unde s anding be e how hese s uc u al di e ences de e mine he unc ional esponse in each case has been a ma e o long- e m in e es . He e we e iew he s uc u al and unc ional p ope ies o h ee dis inc ypes o synapses: he neu omuscula junc ion (NMJ; a gian , highly eliable synapse ha mus exocy ose a la ge numbe o quan a wi h each s imulus o gua an ee exci a ion o he pos synap ic cell), he hippocampal exci a o y small synapse (which mos o en has a single elease si e and a ela i ely small pool o esicles), and he ce ebella mossy ibe -g anule cell synapse (which possesses hund eds o elease si es and is able o ansloca e, dock and p ime esicles a high speed). We will ocus on how he elease appa a us is o ganized in each case, he ela i e amoun o esicula memb ane ha needs o be accommoda ed wi hin he pe iAZ upon s imula ion, he di e en mechanisms o e ie ing he excess o memb ane and inally, how hese ac o s may in luence he unc ioning o he elease si es. Keywo ds: ac i e zone, neu o ansmi e elease, endocy osis, pe iac i e zone, elease si es ORGANIZATION OF THE RELEASE APPARATUS IN SMALL AND LARGE SYNAPSES Synap ic e minals di e in hei s eng h and sho - e m plas ici y, as well as in he size and spa ial o ganiza ion o he sec e o y appa a us, mainly in he dimensions, shape, and amoun o hei Ac i e Zones (AZs), and in he size o hei ecycling pools o synap ic esicles (SVs; A wood and Ka unani hi, 2002; Zhai and Bellen, 2004). Mos e minals in he cen al ne ous sys em (CNS) ha e a small numbe (1–8) o AZs, and he size o he ecycling pool o SVs is no la ge. Fo ins ance, exci a o y ne e e minals in a ea CA1 o he mouse hippocampus ha e a single AZ, abou 10 docked esicles pe AZ, and a ecycling pool o F on ie s in Synap ic Neu oscience | www. on ie sin.o g 1May 2016 | Volume 8 | A icle 12 Cano and Taba es Small and La ge Synapse P ope ies abou 200 SVs (Schiko ski and S e ens, 1997; Mu hy e al., 2001; Rizzoli and Be z, 2005). La ge e minals, on he o he hand, such he a ce ebella mossy ibe –g anule cell synapse, he calyx o Held and he mouse neu omuscula junc ion (NMJ), ha e hund eds o AZs and a la ge ecycling pool o SVs. These s uc u al di e ences a e in acco dance wi h he speci ic unc ional oles o each synapse ype. When he pos synap ic cell esponse is de e mined by he spa ial and empo ally in eg a ed ac i i y o hund eds o small e minals (Figu e 1A), he numbe o quan a eleased pe impulse (quan um con en ) pe e minal is small. Con e sely, when he pos synap ic cell ecei es in o ma ion om only a ew la ge ne e e minals, he sensi i i y and ideli y o he ansmission a e usually e y high. Fo example, in he ce ebella mossy ibe —g anule cell synapse (Figu e 1B), a bu s o ac ion po en ials (APs) in a single mossy ibe bou on could be su icien o gene a e spikes a he g anule cell (Rancz e al., 2007). In his synapse, a la ge ecycling pool o esicles also con ibu es o sus aining ansmission a high equency (Sa iane and Sil e , 2006; Rancz e al., 2007). Finally, when he pos synap ic cell is e y la ge and ecei es only one inpu , he size o he p esynap ic e minal is also big, as a e he numbe o AZs and he ecycling pool o SVs. Typical examples a e he NMJ (Figu e 1C) and he calyx o Held. In e es ingly, e en indi idual neu ons can exhibi la ge di e ences be ween neighbo ing synapses. The hippocampal den a e g anule cell mossy ibe , o example, has 11–18 ela i ely la ge bou ons, each wi h ens o AZs, and in addi ion small e minals a ising om ilopodial ex ensions o he la ge ones (Nicoll and Schmi z, 2005). MEMBRANE LOAD DURING SYNAPTIC ACTIVITY Du ing synap ic ac i i y, esicles used a elease si es a e ansloca ed o he pe iAZ. This p oduces a memb ane load in his compa men , he magni ude o which depends on he du a ion and equency o he s imula ion; ne e heless, i s ela i e impac a ies wi h he AZ o ganiza ion o each e minal. Fo example, a he mouse NMJ (Figu e 1C) om he le a o au is longus (LAL) muscle, a pu e as muscle, a hal -second s imulus ain o 50 APs eleases abou 1700 quan a, he size o eadily eleasable pool (RRP) o SVs in his e minal (Ruiz e al., 2011). Assuming a mean SV diame e o ∼40 nm, he o al memb ane load is ∼8.5 µm2(1700 SVs ×πd2). Howe e , gi en he small size o hei AZs (0.0054 µm2(60 ×90 nm)); Fukunaga e al., 1983; Fukuoka e al., 1987) and he dis ance be ween neighbo ing AZs) ∼0.5 µm; Ruiz e al., 2011), he su ace a ea o each pe iAZ egion (0.5 ×0.5 µm2−0.0054 µm2=0.24 µm2) inc eases only by 4.1% when he wo p ima y docked esicles wi hin each AZ (Nagwaney e al., 2009) use. In ano he la ge synapse, he ce ebella mossy ibe bou on, which also has hund eds o elease si es (Figu e 1B), each one hos ing ∼7–8 docked esicles (Xu-F iedman and Regeh , 2004), he mean a ea o he AZ is abou ou old la ge han in mouse mo o ne e e minals (0.0216 µm2), and he dis ance be ween neighbo ing AZs is ∼0.5 µm (Xu-F iedman e al., 2001; Ruiz e al., 2011). The e o e, in his cen al synapse, i all AZ docked esicles a es use du ing phasic ne e ac i i y, he su ace a ea o each pe iAZ (0.5 ×0.5 µm2−0.0216 µm2=0.2284 µm2) inc eases by ∼16.5%. In con as , in small cen al synapses (Figu e 1A), al hough he quan al con en is much less, a simila numbe o s imuli may p oduce a much la ge ela i e inc emen in he p esynap ic memb ane su ace a ea. Fo example, in CA1 exci a o y hippocampal p esynap ic bou ons, which ha e a mean p esynap ic su ace a ea o a ound 0.2 µm2(Schiko ski and S e ens, 1997), and an AZ a ea o ∼0.027 µm2, i 10 SVs ( he mean size o he RRP) use wi h he p esynap ic memb ane du ing 20 Hz, 2 s s imula ion, he su ace inc eases by ∼0.05 µm2, which ep esen s a ∼29% inc ease o he pe iAZ su ace a ea. The e o e, du ing high equency s imula ion, i he excess o memb ane is no apidly emo ed om he AZ and/o he pe iAZ (Roos and Kelly, 1999) by compensa o y endocy osis, o ansloca ed o dis an egions o la e ission, he ela i e accumula ion o esicula memb ane a he pe iAZ is la ge in small synapses, mainly because hei g ea e numbe o eady- o-go esicles pe AZ, and hei ela i ely smalle pe i-AZ a ea (Figu e 1D). WHEN DOES ENDOCYTOSIS START AND HOW FAST DOES IT GO? Endocy osis is a complex p ocess ha has been s udied mainly by ul as uc u al analysis, elec ical capaci ance measu emen s, and by eal- ime imaging o luo escen molecules associa ed wi h he memb anes. These and o he echniques ha e p o ided e idence ha slow and as modes o endocy osis exis (Figu e 2). Fo example, single SV cla h in-media ed endocy osis is a ela i ely slow p ocess, in he ange o ens o seconds (Heuse and Reese, 1973; G anse h e al., 2006; Balaji e al., 2008; Clay on e al., 2008). In he second mode o endocy osis, he so-called kiss-and- un mode, a e he opening o he usion po e and eleasing o he s o ed ma e ial, he esicle memb ane is apidly eco e ed (∼1s;Cecca elli e al., 1972; Richa ds e al., 2000, 2005; A a anis e al., 2003; Gandhi and S e ens, 2003). Also, he e ie al o a la ge pa ch o memb ane can be achie ed a once by wha i is called bulk endocy osis (Heuse and Reese, 1973; Mille and Heuse , 1984; Hol e al., 2003; Pailla e al., 2003; Clay on e al., 2007; Wu and Wu, 2007; Hayashi e al., 2008). Finally, an ul a as mode o endocy osis, only ac i e a physiological empe a u e, has been desc ibed in which memb ane pa ches, co esponding o he a ea o abou 4 SVs, a e e ie ed wi hin 50–100 ms a e s imula ion (Wa anabe e al., 2013a,b, 2014). Despi e he in o ma ion p o ided by he di e en echniques, he mode o esicle ecycling in each synapse ype is s ill con o e sial. Fo ins ance, he kiss-and- un mode has been desc ibed a he NMJ (Cecca elli e al., 1973), a he calyx o Held (He e al., 2006), and a hippocampal neu ons in cul u e (Ha a a e al., 2006). Ne e heless, in small hippocampal bou ons, many au ho s ha e epo ed ha endocy osis s a s wi h a delay o a ew seconds upon s imula ion and p oceeds slowly. Fo example, F on ie s in Synap ic Neu oscience | www. on ie sin.o g 2May 2016 | Volume 8 | A icle 12 Cano and Taba es Small and La ge Synapse P ope ies FIGURE 1 | S uc u al and unc ional p ope ies o di e en ne e e minals. (A) Ca oon o CA1 exci a o y hippocampal bou ons making con ac s wi h a pos synap ic neu on. (B) Ske ch o ou ce ebella mossy ibe s con ac ing a g anule cell. (C) Illus a ion o a single mo o ne e e minal inne a ing a muscle ibe . (D) Scaled ep esen a ion o he mean sizes o single Ac i e Zones (AZs; pu ple ci cles) and hei co esponding pe i-AZs (g ay ci cles) in h ee synapse ypes ( he neu omuscula junc ion (NMJ), he ce ebella mossy ibe -g anule cell, and he hippocampus), a es (g ay ci cles), and a e usion o all synap ic esicle (SVs) docked a each AZ (b oken line ci cles). No e he simila i y in he pe i-AZ a eas among di e en synapses. (E) Time cons an o luo escence eco e y ( au) s. numbe o s imuli (1, 50 and 100 AP) in mouse mo o ne e e minals (whi e symbols adap ed om Taba es e al., 2007; Ga ield e al., 2009), and in small hippocampal p esynap ic bou ons om neu ons in cul u e, black symbols adap ed om A mb us e e al. (2013). a oom empe a u e, he ime cons an o endocy osis was es ima ed o be 14–16 s a e 1 AP, and he same up o 100 ne e impulses (Figu e 1E;Sanka ana ayanan and Ryan, 2001; Muelle e al., 2004; G anse h e al., 2006; Balaji and Ryan, 2007; Balaji e al., 2008). A he adul mouse NMJ, howe e , he endocy osis ime cons an , measu ed ex i o, was epo ed o be h ee old as e (4–5 s a e 50 APs; Figu e 1E;Taba es e al., 2007; Cano e al., 2012, 2013). When hese measu emen s we e done a physiological empe a u e, he di e ence pe sis ed be ween hese wo synapses. Fo example, a e a single AP he ime cons an o endocy osis is <2 s a he NMJ (Figu e 1E; Ga ield e al., 2009), and be ween 6–15 s a hippocampal small bou ons (Figu e 1E;Balaji e al., 2008; A mb us e e al., 2013). Besides he di e ence in he p e alen mode o endocy osis in dis inc synapses ypes, di e ences in he modula ion o memb ane ecycling a e also p obable. Calcium is a majo modula o o endocy osis in small and la ge synapses. Howe e , depending on he spa io empo al p o ile o he calcium inc emen a elease si es, which in u n, depends on he densi y o calcium channels, he ac i i y o kinases and phospha ases, and he amoun and dis ibu ion o he di e en calcium bu e s, he ou come may a y. E en mo e, he modula ion p ocess is a e y dynamic p ocess in he same synapse. In small hippocampal bou ons, he endocy osis kine ics is accele a ed when s imulus s eng h inc eases om 1 o 25 APs and hen p og essi ely slows o s imulus >25–100 APs (A mb us e e al., 2013). One o he mechanisms by which calcium could accele a e endocy osis is he calcineu in-dependen dephospho yla ion o he p o eins implica ed in endocy osis, known as dephosphins, which include dynamin, synap ojanin, amphiphysin, AP-2, AP-180, among o he s (Ma ks and McMahon, 1998; Cousin and Robinson, 2000). Fo example, he amoun o dynamin dephospho yla ed de e mines, in u n, he in e ac ion o dynamin wi h o he p o eins o he endocy ic machine (Koch e al., 2011; A mb us e e al., 2013; He man and Rosenmund, 2013; Wu e al., 2014). The e o e, he endocy osis speed in di e en e minals could be egula ed no only by he amoun o calcium in lux F on ie s in Synap ic Neu oscience | www. on ie sin.o g 3May 2016 | Volume 8 | A icle 12 Cano and Taba es Small and La ge Synapse P ope ies FIGURE 2 | The successi e s a es o he Release Si es (AZ) and he Pe i-AZ du ing synap ic ac i i y. Upon calcium en y du ing he ac ion po en ial (AP), esicles use a he AZ (pu ple a ea) and elease neu o ansmi e in o he synap ic cle by exocy osis. The SV memb ane componen s a e eco e ed by endocy osis h ough di e en pa hways: kiss-and- un, cla h in-dependen , bulk, and ul a as modes. The clea ing o he spen esicula ma e ial om AZ and pe iAZ a eas is c i ical o he subsequen eloading o new esicles. Clea ance can ake place a he AZ, a he pe iAZ (g ay a ea), o ou side he pe iAZ. Fo simplici y, he shapes o he AZ and pe iAZ a e shown as ec angles and a e no d awn o scaled. The wid hs o he a ows a e p opo ional o he speeds o he p ocess. du ing each AP bu also by he exp ession le el, and spa ial dis ibu ion, o he endocy ic molecula componen s in each synapse ype. ‘‘Ho spo s’’ o endocy ic p o eins nea si es o exocy osis ha e been desc ibed in la ge synapses, o example, in D osophila (Es es e al., 1996; González-Gai án and Jäckle, 1997; Roos and Kelly, 1998), snake (Teng e al., 1999) and mouse (Ga ield e al., 2009) NMJs. In cen al synapses, an en ichmen o endocy ic p o eins a he edges o he AZs is also p obable, as sugges ed by he obse a ion o ul a as endocy osis in his loca ion a hippocampal bou ons (Wa anabe e al., 2013b). The mechanism by which calcium slows endocy osis a e p olonged s imula ion emains unclea . I could be ha calcium inc eases he a e o endocy osis du ing s imula ion un il he capaci y o he endocy ic machine y becomes insu icien (Sanka ana ayanan and Ryan, 2000). A es , he endocy ic machine y is abundan (Roos and Kelly, 1999), bu a e se e al ounds o ac i i y, he consump ion o he endocy ic p o eins may slow he p ocess. The exis ence o a ‘‘cla h in-coa ed eady- o-go pool o esicles’’ a es has been sugges ed a he og NMJ (Mille and Heuse , 1984). The o igin o hese s anded p o ein spo s is, howe e , no clea . Do hey come om used esicles ha ne e los hei iden i y o , al e na i ely, esul a e p o ein in e mixing and so ing? A deg ee o in e mixing be ween esh and old esicle p o eins has been p oposed o occu in hippocampal synapses du ing phasic s imula ion gi en ha s anded, and newly inco po a ed esicle p o eins a e bo h in e nalized du ing compensa o y endocy osis (Fe nandez-Al onso e al., 2006; Wienisch and Klingau , 2006); he longe he s imulus du a ion, he g ea e he in e mixing. Howe e , i is also possible ha endocy osis o bo h new and s anded p o ein pa ches occu in pa allel wi hou p e ious in e mixing (Opazo and Rizzoli, 2010). The de elopmen o new ools will p o ide a deepe unde s anding o he esicle memb ane componen s dynamics du ing phasic and sus ained s imula ion. F on ie s in Synap ic Neu oscience | www. on ie sin.o g 4May 2016 | Volume 8 | A icle 12 Cano and Taba es Small and La ge Synapse P ope ies RELEASE SITE REUSE Du ing ongoing synap ic ansmission, elease si es a e epea edly used. Howe e , has he elease si e a e ac o y pe iod a e use? Knowing he numbe o elease si es a p esynap ic e minal has and he amoun o quan a eleased du ing a ain o s imula ion, i is possible o es ima e he mean minimum numbe o imes a si e is used and he ime in e al be ween uses. Fo example, a he mouse NMJ, a ain o 100 APs a 100 Hz, p oduces he usion o abou 3200 SVs, which ep esen s he size o he whole RRP o esicles (1700) plus abou 1500 mo e (Ruiz e al., 2011). I e oked elease occu s only wi hin he limi s o an AZ, each one hos ing wo elease si es (Nagwaney e al., 2009), and all elease si es a e used a leas once, he ‘‘mean euse index’’ is, in his example, 1.88, esul ing om di iding cumula i e elease by he o al numbe o elease si es (3200/1700). In his case, 88% o si es elease, clea ou o esicula componen s, dock, p ime, and elease again in less han 1 s. In he calyx o Held, a simila ime o e-usage has been es ima ed du ing he i s second o s imula ion a 100 Hz (Nehe , 2010). A highe equencies o s imula ion, he p ocess could be e en as e . Such a apid clea ance o he excess o memb ane a he elease si e could be achie ed ei he by endocy osis in si u (kiss-and- un) o by mo ing he esicula componen s o he pe iAZ om whe e hey a e la e ecycled (Figu e 2). Howe e , when he memb ane load is oo la ge, o example, du ing sus ained high- equency s imula ion, he sys em becomes less e icien . An excessi e memb ane accumula ion a he pe iAZ may in e e e wi h he la e al mo emen o he used memb ane om he AZ o he pe iAZ, e en be o e deple ion o SVs occu s, con ibu ing o sho - e m dep ession (Nehe , 2010; Hua e al., 2013). Rema kably, ansloca ion, docking, and p iming o esicles du ing he pla eau phase ha ollows sho - e m dep ession can also occu e y as du ing sus ained s imula ion. Fo example, in he ce ebella mossy ibe e minal, his p ocess has been sugges ed o ake 12 ms (Sa iane and Sil e , 2006), simila o he elease si e echa ging ime in some ibbon synapses (G iesinge e al., 2005). These obse a ions sugges ha , wi hin a gi en synapse, no all he elease si es has he same capabili y o being eused a high a es o sus ained s imula ion. The basis o his he e ogenei y is no clea . I could be only appa en i some esicles use ou side he ‘‘well s uc u ed’’ elease si es and his speed up he p ocess (Zenisek e al., 2000; Nehe , 2010). Ne e heless, i could also be due o molecula di e ences in he molecula componen s in ol ed in docking/p iming o he esicles, o spa ial a ia ion in he p obabili y o ‘‘in si u’’ endocy osis, and e en o dispa i ies in he eloci y a which dis inc pe iAZs ansloca e esicula componen s o neighbo egions be o e endocy osis. SUMMARY The s uc u al and unc ional p ope ies o p esynap ic e minals a e p incipal de e minan s o he success ul ansmission o in o ma ion in he ne ous sys em. Ne e e minals di e no only in size bu also in he numbe , shape and dimensions o hei AZs and pe iAZs, as well as in he magni ude o he ecycling pool o SVs. La ge e minals ha e hund eds o AZs and elease a la ge numbe o quan a in esponse o s imula ion. Small e minals possess one o ew AZs and elease a low numbe o quan a. The g ea e exocy ic esponse in la ge e minals does no imply a highe load o esicula componen s a he pe iAZs, qui e he con a y, he dis ibu ion o his ma e ial in a la ge numbe o uni s p obably acili a es he managemen o he memb ane excess un il endocy osis akes place. The p e e ed mode o endocy osis used unde each egime o ac i i y in each synapse ype is s ill con o e sial, bu i seems o be as a e b ie s imula ion ains, a leas a physiological empe a u e. Wi h sus ained epe i i e ac i a ion, howe e , endocy osis becomes slowe , p obably due, among o he ac o s, o he sa u a ion o he endocy osis mechanisms and he subsequen accumula ion o esicula memb ane a he pe iAZ. Rema kably, many p esynap ic e minals can sus ain a small and almos cons an amoun o ac i i y (pla eau) upon p olonged high- equency s imula ion, appa en ly suppo ed by a subpopula ion o elease si es ha can ope a e as e han o he s. I he basis o such he e ogenei y is a he le el o he AZ p o eins o he pe iAZs o ganiza ion emains o be de e mined. AUTHOR CONTRIBUTIONS RC and LT concei ed and w o e he manusc ip . ACKNOWLEDGMENTS We a e g a e ul o Bill Be z o discussions and commen s on he manusc ip . This wo k was suppo ed by g an s om he Spanish Minis y o Science and Inno a ion (BFU2013–43763-P) and he Ta iana Pe ez de Guzman Founda ion. REFERENCES A a anis, A. M., Pyle, J. L., Ha a a, N. C., and Tsien, R. W. (2003). Imaging single synap ic esicles unde going epea ed usion e en s: kissing, unning and kissing again. Neu opha macology 45, 797–813. doi: 10.1016/s0028- 3908(03)00310-1 A mb us e , M., Messa, M., Fe guson, S. M., De Camilli, P., and Ryan, T. A. (2013). Dynamin phospho yla ion con ols op imiza ion o endocy osis o b ie ac ion po en ial bu s s. Eli e 2:e00845. doi: 10.7554/eli e.00845 A wood, H. L., and Ka unani hi, S. (2002). Di e si ica ion o synap ic s eng h: p esynap ic elemen s. Na . Re . Neu osci. 3, 497–516. doi: 10.1038/n n876 Balaji, J., A mb us e , M., and Ryan, T. A. (2008). Calcium con ol o endocy ic capaci y a a CNS synapse. J. Neu osci. 28, 6742–6749. doi: 10. 1523/JNEUROSCI.1082-08.2008 Balaji, J., and Ryan, T. A. (2007). Single- esicle imaging e eals ha synap ic esicle exocy osis and endocy osis a e coupled by a single s ochas ic mode. P oc. Na l. Acad. Sci. U S A 104, 20576–20581. doi: 10.1073/pnas.0707574105 Cano, R., Ruiz, R., Shen, C., Taba es, L., and Be z, W. J. (2012). The unc ional landscape o a p esynap ic ne e e minal. Cell Calcium 52, 321–326. doi: 10. 1016/j.ceca.2012.04.012 Cano, R., To es-Beni o, L., Teje o, R., Biea, A. I., Ruiz, R., Be z, W. J., e al. (2013). S uc u al and unc ional ma u a ion o ac i e zones in F on ie s in Synap ic Neu oscience | www. on ie sin.o g 5May 2016 | Volume 8 | A icle 12 Cano and Taba es Small and La ge Synapse P ope ies la ge synapses. Mol. Neu obiol. 47, 209–219. doi: 10.1007/s12035-012- 8347-9 Cecca elli, B., Hu lbu , W. P., and Mau o, A. (1972). Deple ion o esicles om og neu omuscula junc ions by p olonged e anic s imula ion. J. Cell Biol. 54, 30–38. doi: 10.1083/jcb.54.1.30 Cecca elli, B., Hu lbu , W. P., and Mau o, A. (1973). Tu no e o ansmi e and synap ic esicles a he og neu omuscula junc ion. J. Cell Bio. 57, 499–524. doi: 10.1083/jcb.57.2.499 Clay on, E. L., E ans, G. J., and Cousin, M. A. (2007). Ac i i y-dependen con ol o bulk endocy osis by p o ein dephospho yla ion in cen al ne e e minals. J. Physiol. 585, 687–691. doi: 10.1113/jphysiol.2007.137539 Clay on, E. L., E ans, G. J., and Cousin, M. A. (2008). Bulk synap ic esicle endocy osis is apidly igge ed du ing s ong s imula ion. J. Neu osci. 28, 6627–6632. doi: 10.1523/JNEUROSCI.1445-08.2008 Cousin, M. A., and Robinson, P. J. (2000). Ca2+in lux inhibi s dynamin and a es s synap ic esicle endocy osis a he ac i e zone. J. Neu osci. 20, 949–957. Es es, P. S., Roos, J., an de Bliek, A., Kelly, R. B., K ishnan, K. S., and Ramaswami, M. (1996). T a ic o dynamin wi hin indi idual D osophila synap ic bou ons ela i e o compa men -speci ic ma ke s. J. Neu osci. 16, 5443–5456. Fe nandez-Al onso, T., Kwan, R., and Ryan, T. A. (2006). Synap ic esicles in e change hei memb ane p o eins wi h a la ge su ace ese oi du ing ecycling. Neu on 51, 179–186. doi: 10.1016/j.neu on.2006.06.008 Fukunaga, H., Engel, A. G., Lang, B., Newsom-Da is, J., and Vincen , A. (1983). Passi e ans e o Lambe -Ea on myas henic synd ome wi h IgG om man o mouse deple es he p esynap ic memb ane ac i e zones. P oc. Na l. Acad. Sci. U S A 80, 7636–7640. doi: 10.1073/pnas.80.24.7636 Fukuoka, T., Engel, A. G., Lang, B., Newsom-Da is, J., P io , C., and W ay, D. W. (1987). Lambe -Ea on myas henic synd ome: I. Ea ly mo phological e ec s o IgG on he p esynap ic memb ane ac i e zones. Ann. Neu ol. 22, 193–199. doi: 10.1002/ana.410220203 Ga ield, M. A., Taba es, L., and Be z, W. J. (2009). The spa ial pa e n o exocy osis and pos -exocy ic mobili y o synap opHluo in in mouse mo o ne e e minals. J. Physiol. 587, 1187–1200. doi: 10.1113/jphysiol.2008. 166728 Gandhi, S. P., and S e ens, C. F. (2003). Th ee modes o synap ic esicula ecycling e ealed by single- esicle imaging. Na u e 423, 607–613. doi: 10. 1038/na u e01677 González-Gai án, M., and Jäckle, H. (1997). Role o D osophila alpha-adap in in p esynap ic esicle ecycling. Cell 88, 767–776. doi: 10.1016/s0092- 8674(00)81923-6 G anse h, B., Ode ma , B., Royle, S. J., and Lagnado, L. (2006). Cla h in-media ed endocy osis is he dominan mechanism o esicle e ie al a hippocampal synapses. Neu on 51, 773–786. doi: 10.1016/j.neu on.2006.08.029 G iesinge , C. B., Richa ds, C. D., and Ashmo e, J. F. (2005). Fas esicle eplenishmen allows inde a igable signalling a he i s audi o y synapse. Na u e 435, 212–215. doi: 10.1038/na u e03567 Ha a a, N. C., Choi, S., Pyle, J. L., A a anis, A. M., and Tsien, R. W. (2006). F equency-dependen kine ics and p e alence o kiss-and- un and euse a hippocampal synapses s udied wi h no el quenching me hods. Neu on 49, 243–256. doi: 10.1016/j.neu on.2005.12.018 Hayashi, M., Raimondi, A., O’Toole, E., Pa adise, S., Collesi, C., C emona, O., e al. (2008). Cell- and s imulus-dependen he e ogenei y o synap ic esicle endocy ic ecycling mechanisms e ealed by s udies o dynamin 1-null neu ons. P oc. Na l. Acad. Sci. U S A 105, 2175–2180. doi: 10.1073/pnas. 0712171105 He, L., Wu, X. S., Mohan, R., and Wu, L. G. (2006). Two modes o usion po e opening e ealed by cell-a ached eco dings a a synapse. Na u e 444, 102–105. doi: 10.1038/na u e05250 He man, M. A., and Rosenmund, C. (2013). Endocy osis ge s in une wi h ac ion po en ial bu s s. Eli e 2:e01234. doi: 10.7554/eLi e.01234 Heuse , J. E., and Reese, T. S. (1973). E idence o ecycling o synap ic esicle memb ane du ing ansmi e elease a he og neu omuscula junc ion. J. Cell Biol. 57, 315–344. doi: 10.1083/jcb.57.2.315 Hol , M., Cooke, A., Wu, M. M., and Lagnado, L. (2003). Bulk memb ane e ie al in he synap ic e minal o e inal bipola cells. J. Neu osci. 23, 1329–1339. Hua, Y., Woehle , A., Kahms, M., Haucke, V., Nehe , E., and Klingau , J. (2013). Blocking endocy osis enhances sho - e m synap ic dep ession unde condi ions o no mal a ailabili y o esicles. Neu on 80, 343–349. doi: 10.1016/j. neu on.2013.08.010 Koch, D., Spiwoks-Becke , I., Sabano , V., Sinning, A., Dugladze, T., S ellmache , A., e al. (2011). P ope synap ic esicle o ma ion and neu onal ne wo k ac i i y c i ically ely on syndapin I. EMBO J. 30, 4955–4969. doi: 10. 1038/emboj.2011.339 Ma ks, B., and McMahon, H. T. (1998). Calcium igge s calcineu in-dependen synap ic esicle ecycling in mammalian ne e e minals. Cu . Biol. 8, 740–749. doi: 10.1016/s0960-9822(98)70297-0 Mille , T. M., and Heuse , J. E. (1984). Endocy osis o synap ic esicle memb ane a he og neu omuscula junc ion. J. Cell Biol. 98, 685–698. doi: 10.1083/jcb. 98.2.685 Muelle , V. J., Wienisch, M., Neh ing, R. B., and Klingau , J. (2004). Moni o ing cla h in-media ed endocy osis du ing synap ic ac i i y. J. Neu osci. 24, 2004–2012. doi: 10.1523/jneu osci.4080-03.2004 Mu hy, V. N., Schiko ski, T., S e ens, C. F., and Zhu, Y. (2001). Inac i i y p oduces inc eases in neu o ansmi e elease and synapse size. Neu on 32, 673–682. doi: 10.1016/s0896-6273(01)00500-1 Nagwaney, S., Ha low, M. L., Jung, J. H., Szule, J. A., Ress, D., Xu, J., e al. (2009). Mac omolecula connec ions o ac i e zone ma e ial o docked synap ic esicles and p esynap ic memb ane a neu omuscula junc ions o mouse. J. Comp. Neu ol. 513, 457–468. doi: 10.1002/cne.21975 Nehe , E. (2010). Wha is a e-limi ing du ing sus ained synap ic ac i i y: esicle supply o he a ailabili y o elease si es. F on . Synap ic Neu osci. 2:144. doi: 10. 3389/ nsyn.2010.00144 Nicoll, R. A., and Schmi z, D. (2005). Synap ic plas ici y a hippocampal mossy ib e synapses. Na . Re . Neu osci. 6, 863–876. doi: 10.1038/n n1786 Opazo, F., and Rizzoli, S. O. (2010). S udying synap ic esicle pools using pho ocon e sion o s y yl dyes. J. Vis. Exp. 36:1790. doi: 10.3791/1790 Pailla , C., Li, J., Ma hews, G., and S e ling, P. (2003). Endocy osis and esicle ecycling a a ibbon synapse. J. Neu osci. 23, 4092–4099. Rancz, E. A., Ishikawa, T., Duguid, I., Chadde on, P., Mahon, S., and Hausse , M. (2007). High- ideli y ansmission o senso y in o ma ion by single ce ebella mossy ib e bou ons. Na u e 450, 1245–1248. doi: 10.1038/na u e05995 Richa ds, D. A., Bai, J., and Chapman, E. R. (2005). Two modes o exocy osis a hippocampal synapses e ealed by a e o FM1–43 e lux om indi idual esicles. J. Cell Biol. 168, 929–939. doi: 10.1083/jcb.200407148 Richa ds, D. A., Gua imosim, C., and Be z, W. J. (2000). Two endocy ic ecycling ou es selec i ely ill wo esicle pools in og mo o ne e e minals. Neu on 27, 551–559. doi: 10.1016/s0896-6273(00)00065-9 Rizzoli, S. O., and Be z, W. J. (2005). Synap ic esicle pools. Na . Re . Neu osci. 6, 57–69. doi: 10.1038/n n1583 Roos, J., and Kelly, R. B. (1998). Dap160, a neu al-speci ic Eps15 homology and mul iple SH3 domain-con aining p o ein ha in e ac s wi h D osophila dynamin. J. Biol. Chem. 273, 19108–19119. doi: 10.1074/jbc.273.30. 19108 Roos, J., and Kelly, R. B. (1999). The endocy ic machine y in ne e e minals su ounds si es o exocy osis. Cu . Biol. 9, 1411–1414. doi: 10.1016/s0960- 9822(00)80087-1 Ruiz, R., Cano, R., Casanas, J. J., Ga ield, M. A., Be z, W. J., and Taba es, L. (2011). Ac i e zones and he eadily eleasable pool o synap ic esicles a he neu omuscula junc ion o he mouse. J. Neu osci. 31, 2000–2008. doi: 10. 1523/JNEUROSCI.4663-10.2011 Sanka ana ayanan, S., and Ryan, T. A. (2000). Real- ime measu emen s o esicle- SNARE ecycling in synapses o he cen al ne ous sys em. Na . Cell Biol. 2, 197–204. doi: 10.1038/35008615 Sanka ana ayanan, S., and Ryan, T. A. (2001). Calcium accele a es endocy osis o SNAREs a hippocampal synapses. Na . Neu osci. 4, 129–136. doi: 10. 1038/83949 Sa iane, C., and Sil e , R. A. (2006). Fas esicle eloading and a la ge pool sus ain high band wid h ansmission a a cen al synapse. Na u e 439, 983–987. doi: 10.1038/na u e04509 Schiko ski, T., and S e ens, C. F. (1997). Quan i a i e ul as uc u al analysis o hippocampal exci a o y synapses. J. Neu osci. 17, 5858–5867. Taba es, L., Ruiz, R., Lina es-Clemen e, P., Ga ield, M. A., Al a ez de Toledo, G., Fe nandez-Chacon, R., e al. (2007). Moni o ing synap ic unc ion a he neu omuscula junc ion o a mouse exp essing synap op Hluo in. J. Neu osci. 27, 5422–5430. doi: 10.1523/jneu osci.0670-07.2007 F on ie s in Synap ic Neu oscience | www. on ie sin.o g 6May 2016 | Volume 8 | A icle 12 Cano and Taba es Small and La ge Synapse P ope ies Teng, H., Cole, J. C., Robe s, R. L., and Wilkinson, R. S. (1999). Endocy ic ac i e zones: ho spo s o endocy osis in e eb a e neu omuscula e minals. J. Neu osci. 19, 4855–4866. Wa anabe, S., Liu, Q., Da is, M. W., Hollope e , G., Thomas, N., Jo gensen, N. B., e al. (2013a). Ul a as endocy osis a Caeno habdi is elegans neu omuscula junc ions. Eli e 2:e00723. doi: 10.7554/eLi e.00723 Wa anabe, S., Ros , B. R., Camacho-Pe ez, M., Da is, M. W., Sohl- Kielczynski, B., Rosenmund, C., e al. (2013b). Ul a as endocy osis a mouse hippocampal synapses. Na u e 504, 242–247. doi: 10.1038/na u e 12809 Wa anabe, S., T imbuch, T., Camacho-Pe ez, M., Ros , B. R., B okowski, B., Sohl-Kielczynski, B., e al. (2014). Cla h in egene a es synap ic esicles om endosomes. Na u e 515, 228–233. doi: 10.1038/na u e 13846 Wienisch, M., and Klingau , J. (2006). Vesicula p o eins exocy osed and subsequen ly e ie ed by compensa o y endocy osis a e noniden ical. Na . Neu osci. 9, 1019–1027. doi: 10.1038/nn1739 Wu, L. G., Hamid, E., Shin, W., and Chiang, H. C. (2014). Exocy osis and endocy osis: modes, unc ions and coupling mechanisms. Annu. Re . Physiol. 76, 301–331. doi: 10.1146/annu e -physiol-021113-170305 Wu, W., and Wu, L. G. (2007). Rapid bulk endocy osis and i s kine ics o ission po e closu e a a cen al synapse. P oc. Na l. Acad. Sci. U S A 104, 10234–10239. doi: 10.1073/pnas.0611512104 Xu-F iedman, M. A., Ha is, K. M., and Regeh , W. G. (2001). Th ee-dimensional compa ison o ul as uc u al cha ac e is ics a dep essing and acili a ing synapses on o ce ebella Pu kinje cells. J. Neu osci. 21, 6666–6672. Xu-F iedman, M. A., and Regeh , W. G. (2004). S uc u al con ibu ions o sho - e m synap ic plas ici y. Physiol. Re . 84, 69–85. doi: 10.1152/phys e .00016. 2003 Zenisek, D., S eye , J. A., and Alme s, W. (2000). T anspo , cap u e and exocy osis o single synap ic esicles a ac i e zones. Na u e 406, 849–854. doi: 10. 1038/35022500 Zhai, R. G., and Bellen, H. J. (2004). The a chi ec u e o he ac i e zone in he p esynap ic ne e e minal. Physiology (Be hesda) 19, 262–270. doi: 10. 1152/physiol.00014.2004 Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2016 Cano and Taba es. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion and ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) o licenso a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Synap ic Neu oscience | www. on ie sin.o g 7May 2016 | Volume 8 | A icle 12